Compositions and methods for the treatment or prevention of hepatitis B virus infection

ABSTRACT

Disclosed are yeast-based immunotherapeutic compositions, hepatitis B virus (HBV) antigens, and fusion proteins for the treatment and/or prevention of HBV infection and symptoms thereof, as well as methods of using the yeast-based immunotherapeutic compositions, HBV antigens, and fusion proteins for the prophylactic and/or therapeutic treatment of HBV and/or symptoms thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 14/184,481, filed Feb. 19, 2014, now issued U.S. Pat. No. 8,961,988,which is a divisional application of U.S. application Ser. No.13/798,837, filed Mar. 13, 2013, now issued U.S. Pat. No. 8,722,054,which claims the benefit of priority under 35 U.S.C. §120 and is acontinuation of PCT Application No. PCT/US12/24409, filed Feb. 9, 2012,which claims the benefit of priority under 35 U.S.C. §119(e) from eachof U.S. Provisional Application No. 61/442,204, filed Feb. 12, 2011,U.S. Provisional Application No. 61/496,945, filed Jun. 14, 2011, andU.S. Provisional Application No. 61/507,361, filed Jul. 13, 2011. Theentire disclosure of each of U.S. Pat. No. 8,961,988, U.S. Pat. No.8,722,054, PCT Application No. PCT/US12/24409, U.S. ProvisionalApplication No. 61/442,204, U.S. Provisional Application No. 61/496,945,and U.S. Provisional Application No. 61/507,361 is incorporated hereinby reference.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing submitted electronically asa text file by EFS-Web. The text file, named “3923-32-PCT_ST25”, has asize in bytes of 476 KB, and was recorded on Feb. 7, 2012. Theinformation contained in the text file is incorporated herein byreference in its entirety pursuant to 37 CFR §1.52(e)(5).

FIELD OF THE INVENTION

The present invention generally relates to immunotherapeuticcompositions and methods for preventing and/or treating hepatitis Bvirus (HBV) infection.

BACKGROUND OF THE INVENTION

Hepatitis B virus (HBV) is a member of the hepadnavirus family and is acausative agent of acute and chronic hepatitis worldwide. HBV epidemicshave been prevalent in Asia and Africa, and HBV infection is endemic inChina (Williams, R. (2006), “Global challenges in liver disease”,Hepatology (Baltimore, Md.) 44 (3): 521-526). More than 2 billion peoplehave been infected with the virus, and it is estimated that there are350 million chronically HBV-infected individuals worldwide (“HepatitisB”, World Health Organization, 2009; “FAQ About Hepatitis B”, StanfordSchool of Medicine. 2008-07-10). Routes of infection are through bloodand bodily fluid contact, including blood transfusions and IV drug use,sexual transmission, bites and lesions, and vertical transmission (e.g.,childbirth).

HBV is found as one of four major serotypes (adr, adw, ayr, ayw) thatare determined based on antigenic epitopes within its envelope proteins.There are eight different genotypes (A-H) based on the nucleotidesequence variations in the genome. Genotype differences impact diseaseseverity, disease course and likelihood of complications, response totreatment and possibly response to vaccination (Kramvis et al., (2005),Vaccine 23 (19): 2409-2423; Magnius and Norder, (1995), Intervirology 38(1-2): 24-34).

The clinical incubation period for HBV is usually 2-3 months;approximately two thirds of those acutely infected are asymptomatic orhave mild, subclinical symptoms. The remaining one third of acutelyinfected individuals may experience jaundice, inflammation of the liver,vomiting, aches and/or mild fever, but the disease is eventuallyresolved in most adults and rarely leads to liver failure. Indeed,approximately 95% of adults recover completely from HBV infection and donot become chronically infected. However, approximately 90% of infantsand 25%-50% of children aged 1-5 years will remain chronically infectedwith HBV (Centers for Disease Control and Prevention as of September2010). Approximately 25% of those who become chronically infected duringchildhood and 15% of those who become chronically infected afterchildhood die prematurely from cirrhosis or hepatocellular carcinoma,and the majority of chronically infected individuals remain asymptomaticuntil onset of cirrhosis or end-stage liver disease (CDC as of September2010). 1 million deaths per year worldwide (about 2000-4000 deaths peryear in the U.S.) result from chronic HBV infection. Chronicallyinfected individuals have elevated serum alanine aminotransferase (ALT)levels (a marker of liver damage), liver inflammation and/or fibrosisupon liver biopsy. For those patients who develop cirrhosis, the 5 yearsurvival rate is about 50%.

HBV infection and its treatment are typically monitored by the detectionof viral antigens and/or antibodies against the antigens. Upon infectionwith HBV, the first detectable antigen is the hepatitis B surfaceantigen (HBsAg), followed by the hepatitis B “e” antigen (HBeAg).Clearance of the virus is indicated by the appearance of IgG antibodiesin the serum against HBsAg and/or against the core antigen (HBcAg), alsoknown as seroconversion. Numerous studies indicate that viralreplication, the level of viremia and progression to the chronic statein HBV-infected individuals are influenced directly and indirectly byHBV-specific cellular immunity mediated by CD4⁺ helper (T_(H)) and CD8⁺cytotoxic T lymphocytes (CTLs). Patients progressing to chronic diseasetend to have absent, weaker, or narrowly focused HBV-specific T cellresponses as compared to patients who clear acute infection. See, e.g.,Chisari, 1997, J Clin Invest 99: 1472-1477; Maini et al., 1999,Gastroenterology 117:1386-1396; Rehermann et al., 2005, Nat Rev Immunol2005; 5:215-229; Thimme et al., 2001, J Virol 75: 3984-3987; Urbani etal., 2002, J Virol 76: 12423-12434; Wieland and Chisari, 2005, J Virol79: 9369-9380; Webster et al., 2000, Hepatology 32:1117-1124; Penna etal., 1996, J Clin Invest 98: 1185-1194; Sprengers et al., 2006, JHepatol 2006; 45: 182-189.

Vaccines for the prevention of HBV have been commercially availablesince the early 1980's. Current commercial vaccines are non-infectious,subunit viral vaccines providing purified recombinant hepatitis B virussurface antigen (HBsAg), and can be administered beginning at birth. Thevaccines have been effective at reducing the incidence of infection incountries where the vaccine is routinely administered. While a fewimmunotherapeutics are in development, including various HBV protein orepitope vaccines and cytokines, there are currently no approvedimmunotherapeutics for the treatment of active HBV infection in theUnited States.

Current standard of care (SOC) therapy for HBV infection includesprimarily antiviral drugs, such as tenofovir (VIREAD®), lamivudine(EPIVIR®), adefovir (HEPSERA®), telbivudine (TYZEKA®) and entecavir(BARACLUDE®), as well as interferon-α2a and pegylated interferon-α2a(PEGASYS®). These drugs, and particularly the antiviral drugs, aretypically administered for long periods of time (e.g., daily or weeklyfor one to five years or longer), and although they slow or stop viralreplication, they typically do not provide a complete “cure” oreradication of the virus. Interferon-based approaches are toxic and havemodest remission rates. The antiviral therapies inhibit viralreplication and are better tolerated than interferon, but as mentionedabove, these drugs typically do not provide a complete viral cure, andin some cases long term remission rates are not achieved. Moreover, insome cases, development of drug resistance ensues. For example,lamivudine is a potent oral antiviral that inhibits HBV reversetranscriptase (Pol). As lamivudine is well tolerated, and because it isnow a generic drug, lamivudine is an option for HBV antiviral therapy indeveloping countries. However, a 20% annual viral resistance rate frompoint mutations in the Pol sequence limits the utility of lamivudine forHBV. Moreover, response to current anti-viral and interferon treatmentis differently effective among HBV genotypes (Cao, World Journal ofGastroenterology 2009; 15(46):5761-9) and in some patients, because thehepatitis B virus DNA can persist in the body even after infectionclears, reactivation of the virus can occur over time.

Accordingly, while standard of care (SOC) therapy provides the bestcurrently approved treatment for patients suffering from chronic HBV,the length of time for therapy and the significant adverse effects ofthe regimens can lead to noncompliance, dose reduction, and treatmentdiscontinuation, combined with viral escape, reactivation of the virus,and patients who still fail to respond or sustain response to therapy.Therefore, there remains a need in the art for improved therapeutictreatments for HBV infection.

SUMMARY OF THE INVENTION

One embodiment of the invention relates to an immunotherapeuticcomposition for the treatment and/or prevention of hepatitis B virus(HBV) infection and/or a symptom of HBV infection. The immunotherapeuticcomposition comprises: (a) a yeast vehicle; and (b) one or more HBVantigens. In one aspect, the HBV antigens are provided as one or morefusion proteins, although single protein HBV antigens may also beprovided. The HBV antigens consist of: (i) an HBV surface antigencomprising at least one immunogenic domain of a full-length HBV large(L), medium (M) and/or small (S) surface antigen; (ii) an HBV polymeraseantigen comprising at least one immunogenic domain of a full-length HBVpolymerase or domain thereof (e.g., a reverse transcriptase (RT)domain); (iii) an HBV core antigen or HBV e-antigen comprising at leastone immunogenic domain of a full-length HBV core protein and/or afull-length HBV e-antigen, respectively; and/or (iv) an HBV X antigencomprising at least one immunogenic domain of a full-length HBV Xantigen. The composition elicits an HBV-specific immune response againstone or more HBV antigens in the composition and/or against one or moreantigens in a hepatitis B virus that has infected, or may infect, anindividual.

In any of the embodiments of the invention described herein, includingany embodiment related to an immunotherapeutic composition, HBV antigen,fusion protein or use of such composition, HBV antigen or fusionprotein, in one aspect, the amino acid sequence of the HBV large surfaceantigen (L) can include, but is not limited to, an amino acid sequencerepresented by SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:11, SEQ ID NO:15, SEQID NO:19, SEQ ID NO:23, SEQ DI NO:27 or SEQ ID NO:31, or a correspondingsequence from another HBV strain/isolate. The amino acid sequence of HBVpolymerase can include, but is not limited to, an amino acid sequencerepresented by SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:14, SEQID NO:18, SEQ ID NO:22, SEQ ID NO:26 or SEQ ID NO:30, a domain of thesesequences, such as the reverse transcriptase (RT) domain, or acorresponding sequence from another HBV strain/isolate. The amino acidsequence of HBV precore protein, which includes both HBV core proteinsequence and HBV e-antigen sequence, can include, but is not limited to,an amino acid sequence represented by SEQ ID NO:1, SEQ ID NO:5, SEQ IDNO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21, SEQ ID NO:25, or SEQ IDNO:29, or a corresponding sequence from another HBV strain/isolate. Theamino acid sequence of an HBV X antigen can include, but is not limitedto, an amino acid sequence represented by SEQ ID NO:4, SEQ ID NO:8, SEQID NO:12, SEQ ID NO:16, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:28, or SEQID NO:32, or a corresponding sequence from another HBV strain/isolate.

In any of the embodiments of the invention described herein, includingany embodiment related to an immunotherapeutic composition, HBV antigen,fusion protein or use of such composition, HBV antigen or fusionprotein, in one aspect, an amino acid of an HBV surface antigen usefulas an HBV antigen or in a fusion protein or an immunotherapeuticcomposition of the invention can include, but is not limited to, SEQ IDNO:3, SEQ ID NO:7, SEQ ID NO:11, positions 21-47 of SEQ ID NO:11,positions 176-400 of SEQ ID NO:11, SEQ ID NO:15, SEQ ID NO:19, SEQ IDNO:23, SEQ ID NO:27, SEQ ID NO:31, positions 9-407 of SEQ ID NO:34,positions 6-257 of SEQ ID NO:36, positions 6-257 of SEQ ID NO:41,positions 92-343 of SEQ ID NO:92, positions 90-488 of SEQ ID NO:93, SEQID NO:97, positions 90-338 of SEQ ID NO:101, positions 7-254 of SEQ IDNO:102, positions 1-249 of SEQ ID NO:107, positions 1-249 of SEQ IDNO:108, positions 1-249 of SEQ ID NO:109, positions 1-249 of SEQ IDNO:110, positions 1-399 of SEQ ID NO:112, positions 1-399 of SEQ IDNO:114, or positions 1-399 of SEQ ID NO:116, positions 1-399 of SEQ IDNO:118, positions 1-399 of SEQ ID NO:120, positions 1-399 of SEQ IDNO:122, positions 1-399 of SEQ ID NO:124, positions 1-399 of SEQ IDNO:126, positions 231-629 of SEQ ID NO:128, positions 63-461 of SEQ IDNO:130, positions 289-687 of SEQ ID NO:132, positions 289-687 of SEQ IDNO:134, or a corresponding sequence from a different HBV strain.

In any of the embodiments of the invention described herein, includingany embodiment related to an immunotherapeutic composition, HBV antigen,fusion protein or use of such composition, HBV antigen or fusionprotein, in one aspect, an amino acid of an HBV polymerase antigenuseful as an HBV antigen or in a fusion protein or an immunotherapeuticcomposition of the invention can include, but is not limited to,positions 383-602 of SEQ ID NO:2, positions 381-600 of SEQ ID NO:6,positions 381-600 of SEQ ID NO:10, positions 453 to 680 of SEQ ID NO:10,positions 370-589 of SEQ ID NO:14, positions 380-599 of SEQ ID NO:18,positions 381-600 of SEQ ID NO:22, positions 380-599 of SEQ ID NO:26,positions 381-600 of SEQ ID NO:30, positions 260 to 604 of SEQ ID NO:36,positions 7-351 of SEQ ID NO:38, positions 7-351 of SEQ ID NO:40, 260 to604 of SEQ ID NO:41, positions 346 to 690 of SEQ ID NO:92, positions90-434 of SEQ ID NO:94, SEQ ID NO:98, positions 339 to 566 of SEQ IDNO:101, positions 255 to 482 of SEQ ID NO:102, positions 250-477 of SEQID NO:107, positions 250-477 of SEQ ID NO:108, positions 250-477 of SEQID NO:109, positions 250-477 of SEQ ID NO:110, positions 582 to 809 ofSEQ ID NO:120, positions 582 to 809 of SEQ ID NO:124, positions 642 to869 of SEQ ID NO:126, positions 1 to 228 of SEQ ID NO:128, positions 1to 228 of SEQ ID NO:132, positions 61 to 288 of SEQ ID NO:134, or acorresponding sequence from a different HBV strain.

In any of the embodiments of the invention described herein, includingany embodiment related to an immunotherapeutic composition, HBV antigen,fusion protein or use of such composition, HBV antigen or fusionprotein, in one aspect, an amino acid of an HBV core antigen useful asan HBV antigen or in a fusion protein or an immunotherapeuticcomposition of the invention can include, but is not limited to,positions 31-212 of SEQ ID NO:1, positions 31-212 of SEQ ID NO:5,positions 31-212 of SEQ ID NO:9, positions 37 to 188 of SEQ ID NO:9,positions 31-212 of SEQ ID NO:13, positions 31-212 of SEQ ID NO:17,positions 31-212 of SEQ ID NO:21, positions 14-194 of SEQ ID NO:25,positions 31-212 of SEQ ID NO:29, positions 408-589 of SEQ ID NO:34,positions 605 to 786 of SEQ ID NO:36, positions 352-533 of SEQ ID NO:38,positions 160-341 of SEQ ID NO:39, positions 605-786 of SEQ ID NO:41,positions 691-872 of SEQ ID NO:92, positions 90-271 of SEQ ID NO:95, SEQID NO:99, positions 567 to 718 of SEQ ID NO:101, positions 483 to 634 ofSEQ ID NO:102, positions 2-183 of SEQ ID NO:105, positions 184-395 ofSEQ ID NO:105, positions 396-578 of SEQ ID NO:105, positions 579-761 ofSEQ ID NO:105, positions 2-183 of SEQ ID NO:106, 338-520 of SEQ IDNO:106, positions 478-629 of SEQ ID NO:107, positions 478-629 of SEQ IDNO:108, positions 478-629 of SEQ ID NO:109, positions 478-629 of SEQ IDNO:110, positions 400-581 of SEQ ID NO:112, positions 400-581 of SEQ IDNO:114, positions 400-581 of SEQ ID NO:116, positions 400-581 of SEQ IDNO:118, positions 400 to 581 of SEQ ID NO:120, positions 400 to 581 ofSEQ ID NO:122, positions 400 to 581 of SEQ ID NO:124, positions 400 to581 of SEQ ID NO:126, positions 630 to 811 of SEQ ID NO:128, positions462 to 643 of SEQ ID NO:130, positions 688 to 869 of SEQ ID NO:132,positions 688 to 869 of SEQ ID NO:134, or a corresponding sequence froma different HBV strain.

In any of the embodiments of the invention described herein, includingany embodiment related to an immunotherapeutic composition, HBV antigen,fusion protein or use of such composition, HBV antigen or fusionprotein, in one aspect, an amino acid of an HBV X antigen useful as anHBV antigen or in a fusion protein or an immunotherapeutic compositionof the invention can include, but is not limited to, SEQ ID NO:4, SEQ IDNO:8, SEQ ID NO:12, positions 2 to 154 of SEQ ID NO:12, SEQ ID NO:16,SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:32, positions 52-68followed by positions 84-126 of SEQ ID NO:4, positions 52-68 followed bypositions 84-126 of SEQ ID NO:8, positions 52-68 followed by positions84-126 of SEQ ID NO:12, positions 52-68 followed by positions 84-126 ofSEQ ID NO:16, positions 52-68 followed by positions 84-126 of SEQ IDNO:20, positions 52-68 followed by positions 84-126 of SEQ ID NO:24,positions 52-68 followed by positions 84-126 of SEQ ID NO:28, positions52-68 followed by positions 84-126 of SEQ ID NO:32, positions 787 to 939of SEQ ID NO:36, positions 7-159 of SEQ ID NO:39, positions 873-1025 ofSEQ ID NO:92, positions 90-242 of SEQ ID NO:96, SEQ ID NO:100, positions719-778 of SEQ ID NO:101, positions 635-694 of SEQ ID NO:102, positions184-337 of SEQ ID NO:106, positions 521-674 of SEQ ID NO:106, positions630-689 of SEQ ID NO:107, positions 630-689 of SEQ ID NO:108, positions630-689 of SEQ ID NO:109, positions 630-689 of SEQ ID NO:110, positions582-641 of SEQ ID NO:122, positions 810-869 of SEQ ID NO:124, positions582-641 of SEQ ID NO:126, positions 1-60 of SEQ ID NO:130, positions 229to 288 of SEQ ID NO:132, positions 1 to 60 of SEQ ID NO:134, or acorresponding sequence from a different HBV strain.

In one embodiment, the present invention includes an immunotherapeuticcomposition comprising: (a) a yeast vehicle; and (b) a fusion proteincomprising HBV antigens, wherein the HBV antigens consist of: (i) an HBVX antigen comprising at least one immunogenic domain of a full-lengthHBV X antigen; (ii) an HBV surface antigen comprising at least oneimmunogenic domain of a full-length HBV large surface antigen (L), and;(iii) an HBV core antigen comprising at least one immunogenic domain ofa full-length HBV core protein. In one aspect of this embodiment, theimmunotherapeutic composition comprises: (a) a yeast vehicle; and (b) afusion protein comprising HBV antigens, wherein the HBV antigens consistof: (i) an HBV X antigen having an amino acid sequence that is at least80% identical to positions 52 to 126 of a full-length HBV X antigen;(ii) an HBV surface antigen having an amino acid sequence that is atleast 95% identical to an amino acid sequence of a full-length HBV largesurface antigen (L), and; (iii) an HBV core antigen having an amino acidsequence that is at least 95% identical to an amino acid sequence of afull-length HBV core protein. The composition elicits an HBV-specificimmune response.

In one aspect of this embodiment of the invention, the amino acidsequence of HBV X antigen is at least 95% identical, or at least 96%identical, or at least 97% identical, or at least 98% identical, or atleast 99% identical, or is identical, to an amino acid sequence selectedfrom: positions 1-60 of SEQ ID NO:130, positions 630-689 of SEQ IDNO:110, positions 582-641 of SEQ ID NO:122, positions 630-689 of SEQ IDNO:107, positions 630-689 of SEQ ID NO:108, positions 630-689 of SEQ IDNO:109, positions 52-68 followed by positions 84-126 of SEQ ID NO:4,positions 52-68 followed by positions 84-126 of SEQ ID NO:8, positions52-68 followed by positions 84-126 of SEQ ID NO:12, positions 52-68followed by positions 84-126 of SEQ ID NO:16, positions 52-68 followedby positions 84-126 of SEQ ID NO:20, positions 52-68 followed bypositions 84-126 of SEQ ID NO:24, positions 52-68 followed by positions84-126 of SEQ ID NO:28, positions 52-68 followed by positions 84-126 ofSEQ ID NO:32, SEQ ID NO:100, positions 719-778 of SEQ ID NO:101,positions 635-694 of SEQ ID NO:102, positions 810-869 of SEQ ID NO:124,positions 582-641 of SEQ ID NO:126, positions 229 to 288 of SEQ IDNO:132, positions 1 to 60 of SEQ ID NO:134, or a corresponding sequencefrom a different HBV strain. In one aspect, the amino acid sequence ofHBV X antigen is selected from: positions 1-60 of SEQ ID NO:130,positions 630-689 of SEQ ID NO:110, positions 582-641 of SEQ ID NO:122,positions 630-689 of SEQ ID NO:109, positions 630-689 of SEQ ID NO:108,positions 630-689 of SEQ ID NO:107, SEQ ID NO:100, or a correspondingsequence from a different HBV strain.

In one aspect of this embodiment of the invention, the amino acidsequence of the HBV surface antigen is at least 95% identical, or atleast 96% identical, or at least 97% identical, or at least 98%identical, or at least 99% identical, or is identical, to an amino acidsequence selected from: positions 63-461 of SEQ ID NO:130, positions1-399 of SEQ ID NO:118, positions 1-399 of SEQ ID NO:122, positions9-407 of SEQ ID NO:34, positions 1-399 of SEQ ID NO:112, positions 1-399of SEQ ID NO:114, positions 1-399 of SEQ ID NO:116, SEQ ID NO:3, SEQ IDNO:7, SEQ ID NO:11, SEQ ID NO:15, SEQ ID NO:19, SEQ ID NO:23, SEQ IDNO:27, SEQ ID NO:31, positions 90-488 of SEQ ID NO:93, positions 1-399of SEQ ID NO:120, positions 1-399 of SEQ ID NO:124, positions 1-399 ofSEQ ID NO:126, positions 231-629 of SEQ ID NO:128, positions 289-687 ofSEQ ID NO:132, positions 289-687 of SEQ ID NO:134, or a correspondingsequence from a different HBV strain. In one aspect, the amino acidsequence of the HBV surface antigen is selected from: positions 63-461of SEQ ID NO:130, positions 1-399 of SEQ ID NO:118, positions 1-399 ofSEQ ID NO:122, positions 9-407 of SEQ ID NO:34, positions 1-399 of SEQID NO:112, positions 1-399 of SEQ ID NO:114, positions 1-399 of SEQ IDNO:116, or a corresponding sequence from a different HBV strain.

In one aspect of this embodiment of the invention, the amino acidsequence of the HBV core antigen is at least 95% identical, or at least96% identical, or at least 97% identical, or at least 98% identical, orat least 99% identical, or is identical, to an amino acid sequenceselected from: positions 462 to 643 of SEQ ID NO:130, positions 400-581of SEQ ID NO:118, positions 400 to 581 of SEQ ID NO:122, positions408-589 of SEQ ID NO:34, positions 400-581 of SEQ ID NO:112, positions400-581 of SEQ ID NO:114, positions 400-581 of SEQ ID NO:116, positions31-212 of SEQ ID NO:1, positions 31-212 of SEQ ID NO:5, positions 31-212of SEQ ID NO:9, positions 31-212 of SEQ ID NO:13, positions 31-212 ofSEQ ID NO:17, positions 31-212 of SEQ ID NO:21, positions 14-194 of SEQID NO:25, positions 31-212 of SEQ ID NO:29, positions 605 to 786 of SEQID NO:36, positions 352-533 of SEQ ID NO:38, positions 160-341 of SEQ IDNO:39, positions 605-786 of SEQ ID NO:41, positions 691-872 of SEQ IDNO:92, positions 90-271 of SEQ ID NO:95, positions 2-183 of SEQ IDNO:105, positions 184-395 of SEQ ID NO:105, positions 396-578 of SEQ IDNO:105, positions 579-761 of SEQ ID NO:105, positions 2-183 of SEQ IDNO:106, 338-520 of SEQ ID NO:106, positions 400 to 581 of SEQ ID NO:120,positions 400 to 581 of SEQ ID NO:124, positions 400 to 581 of SEQ IDNO:126, positions 630 to 811 of SEQ ID NO:128, positions 688 to 869 ofSEQ ID NO:132, positions 688 to 869 of SEQ ID NO:134, or a correspondingsequence from a different HBV strain. In one aspect, the amino acidsequence of the HBV core antigen is selected from: positions 462 to 643of SEQ ID NO:130, positions 400-581 of SEQ ID NO:118, positions 400 to581 of SEQ ID NO:122, positions 408-589 of SEQ ID NO:34, positions400-581 of SEQ ID NO:116, positions 400-581 of SEQ ID NO:112, positions400-581 of SEQ ID NO:114, or a corresponding sequence from a differentHBV strain.

In one aspect of this embodiment of the invention, the HBV antigens arearranged in the following order, from N- to C-terminus, in the fusionprotein: HBV X antigen, HBV surface antigen, HBV core antigen. In oneaspect of this embodiment of the invention, the HBV antigens arearranged in the following order, from N- to C-terminus, in the fusionprotein: HBV surface antigen, HBV core antigen, HBV X antigen.

In one aspect of this embodiment of the invention, the fusion proteincomprises an amino acid sequence that is at least 95% identical, or atleast 96% identical, or at least 97% identical, or at least 98%identical, or at least 99% identical, or is identical, to an amino acidsequence selected from SEQ ID NO:130, SEQ ID NO:122, or SEQ ID NO:150.

Yet another embodiment of the invention relates to an immunotherapeuticcomposition comprising: (a) a whole, heat-inactivated yeast fromSaccharomyces cerevisiae; and (b) an HBV fusion protein expressed by theyeast, wherein the fusion protein comprises SEQ ID NO:130.

Another embodiment of the invention relates to an immunotherapeuticcomposition comprising: (a) a whole, heat-inactivated yeast fromSaccharomyces cerevisiae; and (b) an HBV fusion protein expressed by theyeast, wherein the fusion protein comprises SEQ ID NO:150.

Yet another embodiment of the invention relates to an immunotherapeuticcomposition comprising: (a) a whole, heat-inactivated yeast fromSaccharomyces cerevisiae; and (b) an HBV fusion protein expressed by theyeast, wherein the fusion protein comprises SEQ ID NO:122. In oneaspect, the fusion protein is a single polypeptide with the followingsequences fused in frame from N- to C-terminus: (1) an amino acidsequence of SEQ ID NO:37; (2) a two amino acid linker peptide ofthreonine-serine; (3) an amino acid sequence of SEQ ID NO:122; and (4) ahexahistidine peptide.

In another embodiment of the invention, the immunotherapeuticcomposition includes: (a) a yeast vehicle; and (b) a fusion proteincomprising HBV antigens consisting of: (i) at least one immunogenicdomain of HBV large surface antigen (L) and (ii) at least oneimmunogenic domain of HBV core protein or HBV e-antigen. The compositionelicits an HBV-specific immune response, such as an immune responseagainst HBV large surface antigen (L) and/or HBV core protein or HBVe-antigen.

In one embodiment, the present invention includes an immunotherapeuticcomposition comprising: (a) a yeast vehicle; and (b) a fusion proteincomprising HBV antigens consisting of: (i) an HBV surface antigen havingan amino acid sequence that is at least 95% identical to an amino acidsequence of a full-length HBV large surface antigen (L), and; (ii) anHBV core antigen having an amino acid sequence that is at least 95%identical to an amino acid sequence of a full-length HBV core protein.The composition elicits an HBV-specific immune response. In one aspectof this embodiment, the HBV antigens consist of an amino acid sequencecomprising at least 95% of a full-length HBV large surface antigen (L)fused to an amino acid sequence comprising at least 95% of a full-lengthHBV core protein or HBV e-antigen. In one aspect of this embodiment, theHBV antigens consist of an amino acid sequence comprising at least 95%of a full-length HBV large surface antigen (L) fused to the N-terminusof an amino acid sequence comprising at least 95% of a full-length HBVcore protein. In one aspect, the HBV antigens consist of: amino acids 2to 400 of HBV large surface antigen (L); and amino acids 31 to 212 ofthe HBV precore protein comprising HBV core protein and a portion of HBVe-antigen.

In one aspect of this embodiment of the invention, the amino acidsequence of the HBV surface antigen is at least 95% identical, or atleast 96% identical, or at least 97% identical, or at least 98%identical, or at least 99% identical, or is identical, to an amino acidsequence selected from: positions 1-399 of SEQ ID NO:118, positions9-407 of SEQ ID NO:34, positions 1-399 of SEQ ID NO:116, positions 1-399of SEQ ID NO:112, positions 1-399 of SEQ ID NO:114, SEQ ID NO:3 orpositions 2-400 of SEQ ID NO:3, SEQ ID NO:7 or positions 2-400 of SEQ IDNO:7, SEQ ID NO:11 or positions 2-400 of SEQ ID NO:11, SEQ ID NO:15 orpositions 2-389 of SEQ ID NO:15, SEQ ID NO:19 or positions 2-399 of SEQID NO:19, SEQ ID NO:23 or positions 2-400 of SEQ ID NO:23, SEQ ID NO:27or positions 2-399 of SEQ ID NO:27, SEQ ID NO:31 or positions 2-400 ofSEQ ID NO:31, positions 90-488 of SEQ ID NO:93, positions 1-399 of SEQID NO:120, positions 1-399 of SEQ ID NO:122, positions 1-399 of SEQ IDNO:124, positions 1-399 of SEQ ID NO:126, positions 231-629 of SEQ IDNO:128, positions 63-461 of SEQ ID NO:130, positions 289-687 of SEQ IDNO:132, positions 289-687 of SEQ ID NO:134, or a corresponding sequencefrom a different HBV strain. In one aspect, the amino acid sequence ofthe HBV surface antigen is selected from: positions 1-399 of SEQ IDNO:118, positions 9-407 of SEQ ID NO:34, positions 1-399 of SEQ IDNO:112, positions 1-399 of SEQ ID NO:114, positions 1-399 of SEQ IDNO:116, or a corresponding sequence from a different HBV strain.

In one aspect of this embodiment of the invention, the amino acidsequence of the HBV core antigen is at least 95% identical, or at least96% identical, or at least 97% identical, or at least 98% identical, orat least 99% identical, or is identical, to an amino acid sequenceselected from: positions 400-581 of SEQ ID NO:118, positions 408-589 ofSEQ ID NO:34, positions 400-581 of SEQ ID NO:116, positions 400-581 ofSEQ ID NO:112, positions 400-581 of SEQ ID NO:114, positions 31-212 ofSEQ ID NO:1, positions 31-212 of SEQ ID NO:5, positions 31-212 of SEQ IDNO:9, positions 31-212 of SEQ ID NO:13, positions 31-212 of SEQ IDNO:17, positions 31-212 of SEQ ID NO:21, positions 14-194 of SEQ IDNO:25, positions 31-212 of SEQ ID NO:29, positions 605 to 786 of SEQ IDNO:36, positions 352-533 of SEQ ID NO:38, positions 160-341 of SEQ IDNO:39, positions 605-786 of SEQ ID NO:41, positions 691-872 of SEQ IDNO:92, positions 90-271 of SEQ ID NO:95, positions 2-183 of SEQ IDNO:105, positions 184-395 of SEQ ID NO:105, positions 396-578 of SEQ IDNO:105, positions 579-761 of SEQ ID NO:105, positions 2-183 of SEQ IDNO:106, 338-520 of SEQ ID NO:106, positions 400 to 581 of SEQ ID NO:120,positions 400 to 581 of SEQ ID NO:122, positions 400 to 581 of SEQ IDNO:124, positions 400 to 581 of SEQ ID NO:126, positions 630 to 811 ofSEQ ID NO:128, positions 462 to 643 of SEQ ID NO:130, positions 688 to869 of SEQ ID NO:132, positions 688 to 869 of SEQ ID NO:134, or acorresponding sequence from a different HBV strain. In one aspect, theamino acid sequence of the HBV core antigen is selected from: positions400-581 of SEQ ID NO:118, positions 408-589 of SEQ ID NO:34, positions400-581 of SEQ ID NO:116, positions 400-581 of SEQ ID NO:112, positions400-581 of SEQ ID NO:114, or a corresponding sequence from a differentHBV strain.

In one aspect of this embodiment of the invention, the HBV antigensconsist of amino acids 9 to 589 of SEQ ID NO:34, or a correspondingsequence from a different HBV strain. In one aspect, the HBV antigensconsist of an amino acid sequence that is at least 95% identical, or atleast 96% identical, or at least 97% identical, or at least 98%identical, or at least 99% identical, or is identical, to an amino acidsequence selected from: SEQ ID NO:118, SEQ ID NO:116, positions 9-589 ofSEQ ID NO:34, SEQ ID NO:112, SEQ ID NO:114, or a corresponding sequencefor a different HBV strain. In one aspect, the HBV antigens consist of afull-length or near full-length HBV large surface antigen (L) and afull-length or near full-length HBV core protein.

In one aspect of this embodiment of the invention, any of the fusionproteins can include an N-terminal amino acid sequence (appended to theN-terminus of the fusion protein) of SEQ ID NO:37. In another aspect,any of the fusion proteins can include an N-terminal amino acid sequenceselected from SEQ ID NO:89 or SEQ ID NO:90. In one aspect, the fusionprotein comprises an amino acid sequence of SEQ ID NO:151.

Yet another embodiment of the invention relates to an immunotherapeuticcomposition comprising: (a) a whole, heat-inactivated yeast fromSaccharomyces cerevisiae; and (b) an HBV fusion protein expressed by theyeast, wherein the fusion protein comprises SEQ ID NO:118.

Another embodiment of the invention relates to an immunotherapeuticcomposition comprising: (a) a whole, heat-inactivated yeast fromSaccharomyces cerevisiae; and (b) an HBV fusion protein expressed by theyeast, wherein the fusion protein comprises SEQ ID NO:151.

Yet another embodiment of the invention relates to an immunotherapeuticcomposition comprising: (a) a whole, heat-inactivated yeast fromSaccharomyces cerevisiae; and (b) an HBV fusion protein expressed by theyeast, wherein the fusion protein comprises the amino acid sequence ofSEQ ID NO:34.

In another embodiment, the present invention includes animmunotherapeutic composition comprising: (a) a yeast vehicle; and (b) afusion protein comprising HBV antigens. The HBV antigens consist of: (i)an HBV surface antigen consisting of at least one immunogenic domain offull-length HBV large (L), medium (M) or small (S) surface antigen; (ii)an HBV polymerase antigen consisting of at least one immunogenic domainof full-length HBV polymerase or of the reverse transcriptase (RT)domain of HBV polymerase; (iii) an HBV core antigen consisting of atleast one immunogenic domain of full-length HBV core protein or offull-length HBV e-antigen; and (iv) an HBV X antigen consisting of atleast one immunogenic domain of full-length HBV X antigen. Thecomposition elicits an HBV-specific immune response. In one aspect ofthis embodiment, the HBV surface antigen comprises at least oneimmunogenic domain of hepatocyte receptor region of Pre-S1 of the HBVlarge surface antigen (L) and at least one immunogenic domain of HBVsmall surface antigen (S).

In one aspect of this embodiment, the HBV antigens consist of: at least95% of the full-length hepatocyte receptor of Pre-S1 of the HBV largesurface antigen (L), at least 95% of the full-length HBV small surfaceantigen (S), at least 95% of the reverse transcriptase domain of HBVpolymerase, at least 95% of the full-length HBV core protein or HBVe-antigen, and at least 95% of the full-length X antigen. In one aspect,the HBV antigens consist of: an HBV large surface antigen (L) comprisingat least 95% of amino acids 120 to 368 of HBV large surface antigen (L);an RT domain of HBV polymerase comprising at least 95% of amino acids453 to 680 of the RT domain of HBV polymerase; an HBV core proteincomprising at least 95% of amino acids 37 to 188 of HBV core protein;and an HBV X antigen comprising at least 80% of amino acids 52 to 127 ofHBV X antigen. In one aspect, the HBV antigens consist of: amino acids21 to 47 of HBV large surface antigen (L) comprising the hepatocytereceptor domain of Pre-S1; amino acids 176 to 400 of HBV large surfaceantigen (L) comprising HBV small surface antigen (S); amino acids 247 to691 of HBV polymerase comprising the reverse transcriptase domain; aminoacids 31 to 212 of HBV precore protein comprising HBV core protein and aportion of HBV e-antigen; and amino acids 2 to 154 of HBV X antigen. Inone aspect, the HBV antigens consist of: an amino acid sequence at least95% identical to amino acids 120 to 368 of HBV large surface antigen(L); an amino acid sequence at least 95% identical to amino acids 453 to680 of the RT domain of HBV polymerase; an amino acid sequence at least95% identical to amino acids 37 to 188 of HBV core protein; and an aminoacid sequence at least 80% identical to amino acids 52 to 127 of HBV Xantigen. In one aspect, the HBV antigens have been modified toincorporate one or more T cell epitopes set forth in Table 5 andrepresented herein by SEQ ID NOs: 42 to 88 or SEQ ID NOs: 135-140. Inone aspect, the HBV large surface antigen (L) comprises an amino acidsequence of SEQ ID NO:97 or a sequence that is 95% identical to SEQ IDNO:97. In one aspect, the RT domain of an HBV polymerase comprises anamino acid sequence of SEQ ID NO:98 or a sequence that is 95% identicalto SEQ ID NO:98. In one aspect, the HBV core protein comprises an aminoacid sequence of SEQ ID NO:99 or a sequence that is 95% identical to SEQID NO:99. In one aspect, the HBV X antigen comprises an amino acidsequence of SEQ ID NO:100 or a sequence that is 95% identical to SEQ IDNO:100.

In one aspect of this embodiment of the invention, the amino acidsequence of the HBV surface antigen is at least 95% identical to anamino acid sequence of a full-length HBV large surface antigen (L). Inone aspect, the amino acid sequence of the HBV surface antigen is atleast 95% identical, or at least 96% identical, or at least 97%identical, or at least 98% identical, or at least 99% identical, or isidentical, to an amino acid sequence selected from: positions 1-399 ofSEQ ID NO:124, positions 1-399 of SEQ ID NO:126, positions 289-687 ofSEQ ID NO:132, positions 289-687 of SEQ ID NO:134, SEQ ID NO:3, SEQ IDNO:7, SEQ ID NO:11, SEQ ID NO:15, SEQ ID NO:19, SEQ ID NO:23, SEQ IDNO:27, SEQ ID NO:31, positions 9-407 of SEQ ID NO:34, positions 90-488of SEQ ID NO:93, positions 1-399 of SEQ ID NO:112, positions 1-399 ofSEQ ID NO:114, positions 1-399 of SEQ ID NO:116, positions 1-399 of SEQID NO:118, positions 1-399 of SEQ ID NO:120, positions 1-399 of SEQ IDNO:122, positions 231-629 of SEQ ID NO:128, positions 63-461 of SEQ IDNO:130, or a corresponding sequence from a different HBV strain.

In one aspect of this embodiment, the amino acid sequence of the HBVsurface antigen is at least 95% identical, or at least 96% identical, orat least 97% identical, or at least 98% identical, or at least 99%identical, or is identical, to an amino acid sequence selected from: SEQID NO:97, positions 1-249 of SEQ ID NO:107, positions 1-249 of SEQ IDNO:108, positions 1-249 of SEQ ID NO:109, positions 1-249 of SEQ IDNO:110, positions 21-47 of SEQ ID NO:11, positions 176-400 of SEQ IDNO:11, positions 6-257 of SEQ ID NO:36, positions 6-257 of SEQ ID NO:41,positions 92-343 of SEQ ID NO:92, positions 90-338 of SEQ ID NO:101,positions 7-254 of SEQ ID NO:102, or a corresponding sequence from adifferent HBV strain.

In one aspect of this embodiment, the HBV polymerase antigen consists ofat least one immunogenic domain of the RT domain of HBV polymerase. Inone aspect, the amino acid sequence of the HBV polymerase antigen is atleast 95% identical, or at least 96% identical, or at least 97%identical, or at least 98% identical, or at least 99% identical, or isidentical, to an amino acid sequence selected from: SEQ ID NO:98,positions 582 to 809 of SEQ ID NO:124, positions 642 to 869 of SEQ IDNO:126, positions 1 to 228 of SEQ ID NO:132, positions 61 to 288 of SEQID NO:134, positions 250-477 of SEQ ID NO:107, positions 250-477 of SEQID NO:108, positions 250-477 of SEQ ID NO:109, positions 250-477 of SEQID NO:110, positions 383-602 of SEQ ID NO:2, positions 381-600 of SEQ IDNO:6, positions 381-600 of SEQ ID NO:10, positions 453 to 680 of SEQ IDNO:10, positions 370-589 of SEQ ID NO:14, positions 380-599 of SEQ IDNO:18, positions 381-600 of SEQ ID NO:22, positions 380-599 of SEQ IDNO:26, positions 381-600 of SEQ ID NO:30, positions 260 to 604 of SEQ IDNO:36, positions 7-351 of SEQ ID NO:38, positions 7-351 of SEQ ID NO:40,260 to 604 of SEQ ID NO:41, positions 346 to 690 of SEQ ID NO:92,positions 90-434 of SEQ ID NO:94, positions 339 to 566 of SEQ ID NO:101,positions 255 to 482 of SEQ ID NO:102, positions 582 to 809 of SEQ IDNO:120, positions 1 to 228 of SEQ ID NO:128, or a corresponding sequencefrom a different HBV strain.

In one aspect of this embodiment, the amino acid sequence of the HBVcore antigen is at least 95% identical to an amino acid sequence of afull-length HBV core protein. In one aspect, the amino acid sequence ofthe HBV core antigen is at least 95% identical, or at least 96%identical, or at least 97% identical, or at least 98% identical, or atleast 99% identical, or is identical, to an amino acid sequence selectedfrom: positions 400 to 581 of SEQ ID NO:124, positions 400 to 581 of SEQID NO:126, positions 688 to 869 of SEQ ID NO:132, positions 688 to 869of SEQ ID NO:134, positions 408-589 of SEQ ID NO:34, positions 400-581of SEQ ID NO:112, positions 400-581 of SEQ ID NO:114, positions 400-581of SEQ ID NO:116, positions 400-581 of SEQ ID NO:118, positions 31-212of SEQ ID NO:1, positions 31-212 of SEQ ID NO:5, positions 31-212 of SEQID NO:9, positions 31-212 of SEQ ID NO:13, positions 31-212 of SEQ IDNO:17, positions 31-212 of SEQ ID NO:21, positions 14-194 of SEQ IDNO:25, positions 31-212 of SEQ ID NO:29, positions 605 to 786 of SEQ IDNO:36, positions 352-533 of SEQ ID NO:38, positions 160-341 of SEQ IDNO:39, positions 605-786 of SEQ ID NO:41, positions 691-872 of SEQ IDNO:92, positions 90-271 of SEQ ID NO:95, positions 2-183 of SEQ IDNO:105, positions 184-395 of SEQ ID NO:105, positions 396-578 of SEQ IDNO:105, positions 579-761 of SEQ ID NO:105, positions 2-183 of SEQ IDNO:106, 338-520 of SEQ ID NO:106, positions 400 to 581 of SEQ ID NO:120,positions 400 to 581 of SEQ ID NO:122, positions 630 to 811 of SEQ IDNO:128, positions 462 to 643 of SEQ ID NO:130, or a correspondingsequence from a different HBV strain.

In one aspect of this embodiment, the amino acid sequence of the HBVcore antigen is at least 95% identical, or at least 96% identical, or atleast 97% identical, or at least 98% identical, or at least 99%identical, or is identical, to an amino acid sequence selected from:positions SEQ ID NO:99, 37 to 188 of SEQ ID NO:9, positions 567 to 718of SEQ ID NO:101, positions 483 to 634 of SEQ ID NO:102, positions478-629 of SEQ ID NO:107, positions 478-629 of SEQ ID NO:108, positions478-629 of SEQ ID NO:109, positions 478-629 of SEQ ID NO:110, or acorresponding sequence from a different HBV strain.

In one aspect of this embodiment, the HBV X antigen consists of an aminoacid sequence that is at least 95% identical to a full-length HBV Xantigen. In one aspect, the HBV X antigen is at least 95% identical, orat least 96% identical, or at least 97% identical, or at least 98%identical, or at least 99% identical, or is identical, to an amino acidsequence selected from: SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12,positions 2 to 154 of SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:20, SEQ IDNO:24, SEQ ID NO:28, SEQ ID NO:32, positions 787 to 939 of SEQ ID NO:36,positions 7-159 of SEQ ID NO:39, positions 873-1025 of SEQ ID NO:92,positions 90-242 of SEQ ID NO:96, positions 184-337 of SEQ ID NO:106,positions 521-674 of SEQ ID NO:106, or a corresponding sequence from adifferent HBV strain.

In one aspect, the HBV X antigen consists of an amino acid sequence thatis at least 80% identical to positions 52 to 126 of a full-length HBV Xantigen. In one aspect, the amino acid sequence of HBV X antigen is atleast 95% identical, or at least 96% identical, or at least 97%identical, or at least 98% identical, or at least 99% identical, or isidentical, to an amino acid sequence selected from: SEQ ID NO:100,positions 810-869 of SEQ ID NO:124, positions 582-641 of SEQ ID NO:126,positions 229 to 288 of SEQ ID NO:132, positions 1 to 60 of SEQ IDNO:134, positions 630-689 of SEQ ID NO:107, positions 630-689 of SEQ IDNO:108, positions 630-689 of SEQ ID NO:109, positions 630-689 of SEQ IDNO:110, positions 52-68 followed by positions 84-126 of SEQ ID NO:4,positions 52-68 followed by positions 84-126 of SEQ ID NO:8, positions52-68 followed by positions 84-126 of SEQ ID NO:12, positions 52-68followed by positions 84-126 of SEQ ID NO:16, positions 52-68 followedby positions 84-126 of SEQ ID NO:20, positions 52-68 followed bypositions 84-126 of SEQ ID NO:24, positions 52-68 followed by positions84-126 of SEQ ID NO:28, positions 52-68 followed by positions 84-126 ofSEQ ID NO:32, positions 719-778 of SEQ ID NO:101, positions 635-694 ofSEQ ID NO:102, positions 582-641 of SEQ ID NO:122, positions 1-60 of SEQID NO:130, or a corresponding sequence from a different HBV strain.

In one aspect of this embodiment, the HBV antigens have an amino acidsequence that is at least 95% identical, or at least 96% identical, orat least 97% identical, or at least 98% identical, or at least 99%identical, or is identical, to an amino acid sequence selected from:positions 6 to 939 of SEQ ID NO:36, positions 92 to 1025 of SEQ IDNO:92, positions 90 to 778 of SEQ ID NO:101, positions 7 to 694 of SEQID NO:102, or a corresponding sequence from a different HBV strain.

In one aspect of this embodiment, the fusion protein comprises an aminoacid sequence that is at least 95% identical, or at least 96% identical,or at least 97% identical, or at least 98% identical, or at least 99%identical, or is identical, to an amino acid sequence selected from: SEQID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:124,SEQ ID NO:126, SEQ ID NO:132 or SEQ ID NO: 134.

Any of the fusion proteins may, in one aspect, comprise an N-terminalsequence selected from SEQ ID NO:37, SEQ ID NO:89, or SEQ ID NO:90.

In one aspect of this embodiment, the fusion protein comprises an aminoacid sequence that is at least 95% identical, or at least 96% identical,or at least 97% identical, or at least 98% identical, or at least 99%identical, or is identical, to an amino acid sequence selected from: SEQID NO:36, SEQ ID NO:92, SEQ ID NO:101, or SEQ ID NO:102.

Another embodiment of the invention relates to an immunotherapeuticcomposition comprising: (a) a yeast vehicle; and (b) a fusion proteincomprising HBV antigens, wherein the HBV antigens consist of: (i) an HBVsurface antigen consisting of at least one immunogenic domain ofhepatocyte receptor region of Pre-S1 of the HBV large surface antigen(L) and at least one immunogenic domain of HBV small surface antigen(S); (ii) an HBV polymerase antigen consisting of at least oneimmunogenic domain of reverse transcriptase domain of HBV polymerase;and (iii) an HBV core antigen consisting of at least one immunogenicdomain of HBV core protein. The composition elicits an HBV-specificimmune response. In one aspect, the HBV antigens consist of at least 95%of full-length hepatocyte receptor of Pre-S1 of HBV large surfaceantigen (L), at least 95% of full-length HBV small surface antigen, atleast 95% of full-length reverse transcriptase domain of HBV polymerase,and at least 95% of full-length HBV core protein. In one aspect, the HBVantigens consist of at least 95% of full-length HBV large surfaceantigen (L), at least 95% of full-length reverse transcriptase domain ofHBV polymerase, and at least 95% of full-length HBV core protein.

In one aspect of this embodiment, the amino acid sequence of the HBVsurface antigen is at least 95% identical, or at least 96% identical, orat least 97% identical, or at least 98% identical, or at least 99%identical, or is identical, to an amino acid sequence selected from:positions 1-399 of SEQ ID NO:120, positions 231-629 of SEQ ID NO:128,positions 1-399 of SEQ ID NO:112, positions 1-399 of SEQ ID NO:114,positions 1-399 of SEQ ID NO:116, positions 1-399 of SEQ ID NO:118,positions 6-257 of SEQ ID NO:41, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:11,positions 21-47 of SEQ ID NO:11, positions 176-400 of SEQ ID NO:11, SEQID NO:15, SEQ ID NO:19, SEQ ID NO:23, SEQ ID NO:27, SEQ ID NO:31,positions 9-407 of SEQ ID NO:34, positions 6-257 of SEQ ID NO:36,positions 92-343 of SEQ ID NO:92, positions 90-488 of SEQ ID NO:93, SEQID NO:97, positions 90-338 of SEQ ID NO:101, positions 7-254 of SEQ IDNO:102, positions 1-249 of SEQ ID NO:107, positions 1-249 of SEQ IDNO:108, positions 1-249 of SEQ ID NO:109, positions 1-249 of SEQ IDNO:110, positions 1-399 of SEQ ID NO:122, positions 1-399 of SEQ IDNO:124, positions 1-399 of SEQ ID NO:126, positions 63-461 of SEQ IDNO:130, positions 289-687 of SEQ ID NO:132, positions 289-687 of SEQ IDNO:134, or a corresponding sequence from a different HBV strain.

In one aspect of this embodiment of the invention, the amino acidsequence of the HBV polymerase antigen is at least 95% identical, or atleast 96% identical, or at least 97% identical, or at least 98%identical, or at least 99% identical, or is identical, to an amino acidsequence selected from: positions 582 to 809 of SEQ ID NO:120, positions1 to 228 of SEQ ID NO:128, positions 250-477 of SEQ ID NO:107, positions250-477 of SEQ ID NO:108, positions 250-477 of SEQ ID NO:109, positions250-477 of SEQ ID NO:110, 260 to 604 of SEQ ID NO:41, positions 383-602of SEQ ID NO:2, positions 381-600 of SEQ ID NO:6, positions 381-600 ofSEQ ID NO:10, positions 453 to 680 of SEQ ID NO:10, positions 370-589 ofSEQ ID NO:14, positions 380-599 of SEQ ID NO:18, positions 381-600 ofSEQ ID NO:22, positions 380-599 of SEQ ID NO:26, positions 381-600 ofSEQ ID NO:30, positions 260 to 604 of SEQ ID NO:36, positions 7-351 ofSEQ ID NO:38, positions 7-351 of SEQ ID NO:40, positions 346 to 690 ofSEQ ID NO:92, positions 90-434 of SEQ ID NO:94, SEQ ID NO:98, positions339 to 566 of SEQ ID NO:101, positions 255 to 482 of SEQ ID NO:102,positions 582 to 809 of SEQ ID NO:124, positions 642 to 869 of SEQ IDNO:126, positions 1 to 228 of SEQ ID NO:132, positions 61 to 288 of SEQID NO:134, or a corresponding sequence from a different HBV strain.

In one aspect of this embodiment of the invention, the amino acidsequence of the HBV core antigen is at least 95% identical, or at least96% identical, or at least 97% identical, or at least 98% identical, orat least 99% identical, or is identical, to an amino acid sequenceselected from: positions 400 to 581 of SEQ ID NO:120, positions 630 to811 of SEQ ID NO:128, positions 400-581 of SEQ ID NO:112, positions400-581 of SEQ ID NO:114, positions 400-581 of SEQ ID NO:116, positions400-581 of SEQ ID NO:118, positions 605-786 of SEQ ID NO:41, positions31-212 of SEQ ID NO:1, positions 31-212 of SEQ ID NO:5, positions 31-212of SEQ ID NO:9, positions 37 to 188 of SEQ ID NO:9, positions 31-212 ofSEQ ID NO:13, positions 31-212 of SEQ ID NO:17, positions 31-212 of SEQID NO:21, positions 14-194 of SEQ ID NO:25, positions 31-212 of SEQ IDNO:29, positions 408-589 of SEQ ID NO:34, positions 605 to 786 of SEQ IDNO:36, positions 352-533 of SEQ ID NO:38, positions 160-341 of SEQ IDNO:39, positions 691-872 of SEQ ID NO:92, positions 90-271 of SEQ IDNO:95, SEQ ID NO:99, positions 567 to 718 of SEQ ID NO:101, positions483 to 634 of SEQ ID NO:102, positions 2-183 of SEQ ID NO:105, positions184-395 of SEQ ID NO:105, positions 396-578 of SEQ ID NO:105, positions579-761 of SEQ ID NO:105, positions 2-183 of SEQ ID NO:106, 338-520 ofSEQ ID NO:106, positions 478-629 of SEQ ID NO:107, positions 478-629 ofSEQ ID NO:108, positions 478-629 of SEQ ID NO:109, positions 478-629 ofSEQ ID NO:110, positions 400 to 581 of SEQ ID NO:122, positions 400 to581 of SEQ ID NO:124, positions 400 to 581 of SEQ ID NO:126, positions462 to 643 of SEQ ID NO:130, positions 688 to 869 of SEQ ID NO:132,positions 688 to 869 of SEQ ID NO:134, or a corresponding sequence froma different HBV strain.

In one aspect of this embodiment of the invention, the fusion proteinhas an amino acid sequence that is at least 95% identical, or at least96% identical, or at least 97% identical, or at least 98% identical, orat least 99% identical, or is identical, to an amino acid sequenceselected from: SEQ ID NO:120, SEQ ID NO:128, positions 6-786 of SEQ IDNO:41, or SEQ ID NO:41, or a corresponding sequence from a different HBVstrain.

Another embodiment of the invention relates to an immunotherapeuticcomposition comprising: (a) a yeast vehicle; and (b) a fusion proteincomprising HBV antigens, wherein the HBV antigens consist of: (i) an HBVpolymerase antigen consisting of at least one immunogenic domain of thereverse transcriptase (RT) domain of HBV polymerase; and (ii) an HBVcore antigen consisting of at least one immunogenic domain of HBV coreprotein. The composition elicits an HBV-specific immune response. In oneaspect of this embodiment of the invention, the HBV antigens consist of:an amino acid sequence that is at least 95% identical to full-length RTdomain of HBV polymerase and an amino acid sequence that is at least 95%identical to full-length HBV core protein.

In one aspect of this embodiment of the invention, the amino acidsequence of the HBV polymerase antigen is at least 95% identical, or atleast 96% identical, or at least 97% identical, or at least 98%identical, or at least 99% identical, or is identical, to an amino acidsequence selected from: positions 7-351 of SEQ ID NO:38, positions383-602 of SEQ ID NO:2, positions 381-600 of SEQ ID NO:6, positions381-600 of SEQ ID NO:10, positions 453 to 680 of SEQ ID NO:10, positions370-589 of SEQ ID NO:14, positions 380-599 of SEQ ID NO:18, positions381-600 of SEQ ID NO:22, positions 380-599 of SEQ ID NO:26, positions381-600 of SEQ ID NO:30, positions 260 to 604 of SEQ ID NO:36, positions7-351 of SEQ ID NO:40, 260 to 604 of SEQ ID NO:41, positions 346 to 690of SEQ ID NO:92, positions 90-434 of SEQ ID NO:94, SEQ ID NO:98,positions 339 to 566 of SEQ ID NO:101, positions 255 to 482 of SEQ IDNO:102, positions 250-477 of SEQ ID NO:107, positions 250-477 of SEQ IDNO:108, positions 250-477 of SEQ ID NO:109, positions 250-477 of SEQ IDNO:110, positions 582 to 809 of SEQ ID NO:120, positions 582 to 809 ofSEQ ID NO:124, positions 642 to 869 of SEQ ID NO:126, positions 1 to 228of SEQ ID NO:128, positions 1 to 228 of SEQ ID NO:132, positions 61 to288 of SEQ ID NO:134, or a corresponding sequence from a different HBVstrain.

In one aspect of this embodiment of the invention, the amino acidsequence of the HBV core antigen is at least 95% identical, or at least96% identical, or at least 97% identical, or at least 98% identical, orat least 99% identical, or is identical, to an amino acid sequenceselected from: positions 352-533 of SEQ ID NO:38, positions 31-212 ofSEQ ID NO:1, positions 31-212 of SEQ ID NO:5, positions 31-212 of SEQ IDNO:9, positions 37 to 188 of SEQ ID NO:9, positions 31-212 of SEQ IDNO:13, positions 31-212 of SEQ ID NO:17, positions 31-212 of SEQ IDNO:21, positions 14-194 of SEQ ID NO:25, positions 31-212 of SEQ IDNO:29, positions 408-589 of SEQ ID NO:34, positions 605 to 786 of SEQ IDNO:36, positions 160-341 of SEQ ID NO:39, positions 605-786 of SEQ IDNO:41, positions 691-872 of SEQ ID NO:92, positions 90-271 of SEQ IDNO:95, SEQ ID NO:99, positions 567 to 718 of SEQ ID NO:101, positions483 to 634 of SEQ ID NO:102, positions 2-183 of SEQ ID NO:105, positions184-395 of SEQ ID NO:105, positions 396-578 of SEQ ID NO:105, positions579-761 of SEQ ID NO:105, positions 2-183 of SEQ ID NO:106, 338-520 ofSEQ ID NO:106, positions 478-629 of SEQ ID NO:107, positions 478-629 ofSEQ ID NO:108, positions 478-629 of SEQ ID NO:109, positions 478-629 ofSEQ ID NO:110, positions 400-581 of SEQ ID NO:112, positions 400-581 ofSEQ ID NO:114, positions 400-581 of SEQ ID NO:116, positions 400-581 ofSEQ ID NO:118, positions 400 to 581 of SEQ ID NO:120, positions 400 to581 of SEQ ID NO:122, positions 400 to 581 of SEQ ID NO:124, positions−400 to 581 of SEQ ID NO:126, positions 630 to 811 of SEQ ID NO:128,positions 462 to 643 of SEQ ID NO:130, positions 688 to 869 of SEQ IDNO:132, positions 688 to 869 of SEQ ID NO:134, or a correspondingsequence from a different HBV strain.

In one aspect of this embodiment of the invention, the fusion proteinhas an amino acid sequence that is at least 95% identical, or at least96% identical, or at least 97% identical, or at least 98% identical, orat least 99% identical, or is identical, to an amino acid sequence ofSEQ ID NO:38, or a corresponding sequence from a different HBV strain.

Yet another embodiment of the invention relates to an immunotherapeuticcomposition comprising: (a) a yeast vehicle; and (b) a fusion proteincomprising HBV antigens, wherein the HBV antigens consist of: (i) an HBVX antigen consisting of at least one immunogenic domain of HBV Xantigen; and (ii) an HBV core antigen consisting of at least oneimmunogenic domain of HBV core protein. The composition elicits anHBV-specific immune response. In one aspect of this embodiment, the HBVantigens consist of: an amino acid sequence that is at least 95%identical to full-length HBV X antigen and an amino acid sequence thatis at least 95% identical to full-length HBV core protein.

In one aspect of this embodiment of the invention, the amino acidsequence of the HBV core antigen is at least 95% identical, or at least96% identical, or at least 97% identical, or at least 98% identical, orat least 99% identical, or is identical, to an amino acid sequenceselected from: positions 160-341 of SEQ ID NO:39, positions 31-212 ofSEQ ID NO:1, positions 31-212 of SEQ ID NO:5, positions 31-212 of SEQ IDNO:9, positions 37 to 188 of SEQ ID NO:9, positions 31-212 of SEQ IDNO:13, positions 31-212 of SEQ ID NO:17, positions 31-212 of SEQ IDNO:21, positions 14-194 of SEQ ID NO:25, positions 31-212 of SEQ IDNO:29, positions 408-589 of SEQ ID NO:34, positions 605 to 786 of SEQ IDNO:36, positions 352-533 of SEQ ID NO:38, positions 605-786 of SEQ IDNO:41, positions 691-872 of SEQ ID NO:92, positions 90-271 of SEQ IDNO:95, SEQ ID NO:99, positions 567 to 718 of SEQ ID NO:101, positions483 to 634 of SEQ ID NO:102, positions 2-183 of SEQ ID NO:105, positions184-395 of SEQ ID NO:105, positions 396-578 of SEQ ID NO:105, positions579-761 of SEQ ID NO:105, positions 2-183 of SEQ ID NO:106, 338-520 ofSEQ ID NO:106, positions 478-629 of SEQ ID NO:107, positions 478-629 ofSEQ ID NO:108, positions 478-629 of SEQ ID NO:109, positions 478-629 ofSEQ ID NO:110, positions 400-581 of SEQ ID NO:112, positions 400-581 ofSEQ ID NO:114, positions 400-581 of SEQ ID NO:116, positions 400-581 ofSEQ ID NO:118, positions 400 to 581 of SEQ ID NO:120, positions 400 to581 of SEQ ID NO:122, positions 400 to 581 of SEQ ID NO:124, positions−400 to 581 of SEQ ID NO:126, positions 630 to 811 of SEQ ID NO:128,positions 462 to 643 of SEQ ID NO:130, positions 688 to 869 of SEQ IDNO:132, positions 688 to 869 of SEQ ID NO:134, or a correspondingsequence from a different HBV strain.

In one aspect of this embodiment of the invention, the amino acidsequence of the HBV X antigen is at least 95% identical, or at least 96%identical, or at least 97% identical, or at least 98% identical, or atleast 99% identical, or is identical, to an amino acid sequence selectedfrom: positions 7-159 of SEQ ID NO:39, SEQ ID NO:4, SEQ ID NO:8, SEQ IDNO:12, positions 2 to 154 of SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:20,SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:32, positions 52-68 followed bypositions 84-126 of SEQ ID NO:4, positions 52-68 followed by positions84-126 of SEQ ID NO:8, positions 52-68 followed by positions 84-126 ofSEQ ID NO:12, positions 52-68 followed by positions 84-126 of SEQ IDNO:16, positions 52-68 followed by positions 84-126 of SEQ ID NO:20,positions 52-68 followed by positions 84-126 of SEQ ID NO:24, positions52-68 followed by positions 84-126 of SEQ ID NO:28, positions 52-68followed by positions 84-126 of SEQ ID NO:32, positions 787 to 939 ofSEQ ID NO:36, positions 873-1025 of SEQ ID NO:92, positions 90-242 ofSEQ ID NO:96, SEQ ID NO:100, positions 719-778 of SEQ ID NO:101,positions 635-694 of SEQ ID NO:102, positions 184-337 of SEQ ID NO:106,positions 521-674 of SEQ ID NO:106, positions 630-689 of SEQ ID NO:107,positions 630-689 of SEQ ID NO:108, positions 630-689 of SEQ ID NO:109,positions 630-689 of SEQ ID NO:110, positions 582-641 of SEQ ID NO:122,positions 810-869 of SEQ ID NO:124, positions 582-641 of SEQ ID NO:126,positions 1-60 of SEQ ID NO:130, positions 229 to 288 of SEQ ID NO:132,positions 1 to 60 of SEQ ID NO:134, or a corresponding sequence from adifferent HBV strain.

In one aspect of this embodiment of the invention, the fusion proteinhas the amino acid sequence that is at least 95% identical, or at least96% identical, or at least 97% identical, or at least 98% identical, orat least 99% identical, or is identical, to an amino acid sequence toSEQ ID NO:39, or a corresponding sequence from a different HBV strain.

Another embodiment of the invention relates to an immunotherapeuticcomposition comprising: (a) a yeast vehicle; and (b) a fusion proteincomprising an HBV surface antigen consisting of at least one immunogenicdomain of an HBV large surface antigen (L), wherein the compositionelicits an HBV-specific immune response. In one aspect of thisembodiment, the HBV surface antigen consists of at least 95% offull-length HBV large surface antigen (L). In one aspect, the amino acidsequence of the HBV surface antigen is at least 95% identical, or atleast 96% identical, or at least 97% identical, or at least 98%identical, or at least 99% identical, or is identical, to an amino acidsequence selected from: positions 90-488 of SEQ ID NO:93, SEQ ID NO:3,SEQ ID NO:7, SEQ ID NO:11, positions 21-47 of SEQ ID NO:11, positions176-400 of SEQ ID NO:11, SEQ ID NO:15, SEQ ID NO:19, SEQ ID NO:23, SEQID NO:27, SEQ ID NO:31, positions 9-407 of SEQ ID NO:34, positions 6-257of SEQ ID NO:36, positions 6-257 of SEQ ID NO:41, positions 92-343 ofSEQ ID NO:92, positions 90-488 of SEQ ID NO:93, SEQ ID NO:97, positions90-338 of SEQ ID NO:101, positions 7-254 of SEQ ID NO:102, positions1-249 of SEQ ID NO:107, positions 1-249 of SEQ ID NO:108, positions1-249 of SEQ ID NO:109, positions 1-249 of SEQ ID NO:110, positions1-399 of SEQ ID NO:112, positions 1-399 of SEQ ID NO:114, positions1-399 of SEQ ID NO:116, positions 1-399 of SEQ ID NO:118, positions1-399 of SEQ ID NO:120, positions 1-399 of SEQ ID NO:122, positions1-399 of SEQ ID NO:124, positions 1-399 of SEQ ID NO:126, positions231-629 of SEQ ID NO:128, positions 63-461 of SEQ ID NO:130, positions289-687 of SEQ ID NO:132, positions 289-687 of SEQ ID NO:134, or acorresponding sequence from a different HBV strain. In one aspect, thefusion protein has the amino acid sequence that is at least 95%identical, or at least 96% identical, or at least 97% identical, or atleast 98% identical, or at least 99% identical, or is identical, to anamino acid sequence of SEQ ID NO:93, or a corresponding sequence from adifferent HBV strain.

Yet another embodiment of the invention relates to an immunotherapeuticcomposition comprising: (a) a yeast vehicle; and (b) a fusion proteincomprising an HBV polymerase antigen consisting of at least oneimmunogenic domain of a reverse transcriptase domain of HBV polymerase,wherein the composition elicits an HBV-specific immune response. In oneaspect of this embodiment of the invention, the HBV polymerase antigenconsists of at least 95% of full-length reverse transcriptase domain ofHBV polymerase. In one aspect, the amino acid sequence of the HBVpolymerase antigen is at least 95% identical, or at least 96% identical,or at least 97% identical, or at least 98% identical, or at least 99%identical, or is identical, to an amino acid sequence selected from:positions 7-351 of SEQ ID NO:40, positions 90-434 of SEQ ID NO:94,positions 383-602 of SEQ ID NO:2, positions 381-600 of SEQ ID NO:6,positions 381-600 of SEQ ID NO:10, positions 453 to 680 of SEQ ID NO:10,positions 370-589 of SEQ ID NO:14, positions 380-599 of SEQ ID NO:18,positions 381-600 of SEQ ID NO:22, positions 380-599 of SEQ ID NO:26,positions 381-600 of SEQ ID NO:30, positions 260 to 604 of SEQ ID NO:36,positions 7-351 of SEQ ID NO:38, 260 to 604 of SEQ ID NO:41, positions346 to 690 of SEQ ID NO:92, SEQ ID NO:98, positions 339 to 566 of SEQ IDNO:101, positions 255 to 482 of SEQ ID NO:102, positions 250-477 of SEQID NO:107, positions 250-477 of SEQ ID NO:108, positions 250-477 of SEQID NO:109, positions 250-477 of SEQ ID NO:110, positions 582 to 809 ofSEQ ID NO:120, positions 582 to 809 of SEQ ID NO:124, positions 642 to869 of SEQ ID NO:126, positions 1 to 228 of SEQ ID NO:128, positions 1to 228 of SEQ ID NO:132, positions 61 to 288 of SEQ ID NO:134, or acorresponding sequence from a different HBV strain. In one aspect, thefusion protein has the amino acid sequence that is at least 95%identical, or at least 96% identical, or at least 97% identical, or atleast 98% identical, or at least 99% identical, or is identical, to anamino acid sequence of SEQ ID NO:40 or SEQ ID NO:94, or a correspondingsequence from a different HBV strain.

Another embodiment of the invention relates to an immunotherapeuticcomposition comprising: (a) a yeast vehicle; and (b) a fusion proteincomprising an HBV core antigen consisting of at least one immunogenicdomain of an HBV core protein, wherein the composition elicits anHBV-specific immune response. In one aspect of this embodiment of theinvention, the HBV antigens consist of at least 95% of full-length HBVcore protein. In one aspect, the amino acid sequence of the HBV coreantigen is at least 95% identical, or at least 96% identical, or atleast 97% identical, or at least 98% identical, or at least 99%identical, or is identical, to an amino acid sequence selected from:positions 90-271 of SEQ ID NO:95, positions 31-212 of SEQ ID NO:1,positions 31-212 of SEQ ID NO:5, positions 31-212 of SEQ ID NO:9,positions 37 to 188 of SEQ ID NO:9, positions 31-212 of SEQ ID NO:13,positions 31-212 of SEQ ID NO:17, positions 31-212 of SEQ ID NO:21,positions 14-194 of SEQ ID NO:25, positions 31-212 of SEQ ID NO:29,positions 408-589 of SEQ ID NO:34, positions 605 to 786 of SEQ ID NO:36,positions 352-533 of SEQ ID NO:38, positions 160-341 of SEQ ID NO:39,positions 605-786 of SEQ ID NO:41, positions 691-872 of SEQ ID NO:92,SEQ ID NO:99, positions 567 to 718 of SEQ ID NO:101, positions 483 to634 of SEQ ID NO:102, positions 2-183 of SEQ ID NO:105, positions184-395 of SEQ ID NO:105, positions 396-578 of SEQ ID NO:105, positions579-761 of SEQ ID NO:105, positions 2-183 of SEQ ID NO:106, 338-520 ofSEQ ID NO:106, positions 478-629 of SEQ ID NO:107, positions 478-629 ofSEQ ID NO:108, positions 478-629 of SEQ ID NO:109, positions 478-629 ofSEQ ID NO:110, positions 400-581 of SEQ ID NO:112, positions 400-581 ofSEQ ID NO:114, positions 400-581 of SEQ ID NO:116, positions 400-581 ofSEQ ID NO:118, positions 400 to 581 of SEQ ID NO:120, positions 400 to581 of SEQ ID NO:122, positions 400 to 581 of SEQ ID NO:124, positions400 to 581 of SEQ ID NO:126, positions 630 to 811 of SEQ ID NO:128,positions 462 to 643 of SEQ ID NO:130, positions 688 to 869 of SEQ IDNO:132, positions 688 to 869 of SEQ ID NO:134, or a correspondingsequence from a different HBV strain. In one aspect, the protein has theamino acid sequence that is at least 95% identical, or at least 96%identical, or at least 97% identical, or at least 98% identical, or atleast 99% identical, or is identical, to an amino acid sequence of SEQID NO:95, or a corresponding sequence from a different HBV strain.

Yet another embodiment of the invention relates to an immunotherapeuticcomposition comprising: (a) a yeast vehicle; and (b) a fusion proteincomprising an HBV X antigen consisting of at least one immunogenicdomain of a full-length HBV X antigen, wherein the composition elicitsan HBV-specific immune response. In one aspect, the HBV antigen consistsof at least 95% of full-length HBV X antigen. In one aspect, the aminoacid sequence of the HBV X antigen is at least 95% identical, or atleast 96% identical, or at least 97% identical, or at least 98%identical, or at least 99% identical, or is identical, to an amino acidsequence selected from: positions 90-242 of SEQ ID NO:96, SEQ ID NO:4,SEQ ID NO:8, SEQ ID NO:12, positions 2 to 154 of SEQ ID NO:12, SEQ IDNO:16, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:32, positions52-68 followed by positions 84-126 of SEQ ID NO:4, positions 52-68followed by positions 84-126 of SEQ ID NO:8, positions 52-68 followed bypositions 84-126 of SEQ ID NO:12, positions 52-68 followed by positions84-126 of SEQ ID NO:16, positions 52-68 followed by positions 84-126 ofSEQ ID NO:20, positions 52-68 followed by positions 84-126 of SEQ IDNO:24, positions 52-68 followed by positions 84-126 of SEQ ID NO:28,positions 52-68 followed by positions 84-126 of SEQ ID NO:32, positions787 to 939 of SEQ ID NO:36, positions 7-159 of SEQ ID NO:39, positions873-1025 of SEQ ID NO:92, SEQ ID NO:100, positions 719-778 of SEQ IDNO:101, positions 635-694 of SEQ ID NO:102, positions 184-337 of SEQ IDNO:106, positions 521-674 of SEQ ID NO:106, positions 630-689 of SEQ IDNO:107, positions 630-689 of SEQ ID NO:108, positions 630-689 of SEQ IDNO:109, positions 630-689 of SEQ ID NO:110, positions 582-641 of SEQ IDNO:122, positions 810-869 of SEQ ID NO:124, positions 582-641 of SEQ IDNO:126, positions 1-60 of SEQ ID NO:130, positions 229 to 288 of SEQ IDNO:132, positions 1 to 60 of SEQ ID NO:134, or a corresponding sequencefrom a different HBV strain. In one aspect, the protein has the aminoacid sequence that is at least 95% identical, or at least 96% identical,or at least 97% identical, or at least 98% identical, or at least 99%identical, or is identical, to an amino acid sequence of SEQ ID NO:96,or a corresponding sequence from a different HBV strain.

Another embodiment of the invention relates to an immunotherapeuticcomposition comprising any two, three or four of the immunotherapeuticcompositions described above, or elsewhere herein and in particular, anytwo, three, or four of the immunotherapeutic compositions describedabove that relate to single HBV proteins.

Yet another embodiment of the invention relates to an immunotherapeuticcomposition comprising: (a) a yeast vehicle; and (b) a fusion proteincomprising HBV antigens, wherein the HBV antigens consist of at leastone immunogenic domain of two, three or four HBV surface antigenproteins, wherein each of the HBV surface antigen proteins is from adifferent HBV genotype. The composition elicits an HBV-specific immuneresponse.

Yet another embodiment of the invention relates to an immunotherapeuticcomposition comprising: (a) a yeast vehicle; and (b) a fusion proteincomprising HBV antigens, wherein the HBV antigens consist of at leastone immunogenic domain of two, three or four HBV polymerase proteins,wherein each of the HBV polymerase proteins is from a different HBVgenotype. The composition elicits an HBV-specific immune response.

Yet another embodiment of the invention relates to an immunotherapeuticcomposition comprising: (a) a yeast vehicle; and (b) a fusion proteincomprising HBV antigens, wherein the HBV antigens consist of at leastone immunogenic domain of two, three or four HBV X antigens, whereineach of the HBV X antigens is from a different HBV genotype. Thecomposition elicits an HBV-specific immune response.

Yet another embodiment of the invention relates to an immunotherapeuticcomposition comprising: (a) a yeast vehicle; and (b) a fusion proteincomprising HBV antigens, wherein the HBV antigens consist of at leastone immunogenic domain of two, three or four HBV core proteins, whereineach of the HBV core proteins is from a different HBV genotype. Thecomposition elicits an HBV-specific immune response. In one aspect, eachof the HBV core proteins consists of at least 95% of a full-length HBVcore protein. In one aspect, each of the HBV core proteins consists ofamino acids 31 to 212 of HBV core protein. In one aspect, the HBVgenotypes include genotype C, and in one aspect, the HBV genotypesinclude genotype D, and in one aspect, the HBV genotypes includegenotype A, and in one aspect, the HBV genotypes include genotype B. Inone aspect, each of the HBV core proteins consists of amino acids 37 to188 of HBV core protein. In one aspect, the fusion protein comprisesfour HBV core proteins from genotype A, genotype B, genotype C andgenotype D.

In one aspect of this embodiment of the invention, the amino acidsequence of any one or more of the HBV core antigens is at least 95%identical, or at least 96% identical, or at least 97% identical, or atleast 98% identical, or at least 99% identical, or is identical, to anamino acid sequence selected from: positions 90-271 of SEQ ID NO:95,positions 31-212 of SEQ ID NO:1, positions 31-212 of SEQ ID NO:5,positions 31-212 of SEQ ID NO:9, positions 37 to 188 of SEQ ID NO:9,positions 31-212 of SEQ ID NO:13, positions 31-212 of SEQ ID NO:17,positions 31-212 of SEQ ID NO:21, positions 14-194 of SEQ ID NO:25,positions 31-212 of SEQ ID NO:29, positions 408-589 of SEQ ID NO:34,positions 605 to 786 of SEQ ID NO:36, positions 352-533 of SEQ ID NO:38,positions 160-341 of SEQ ID NO:39, positions 605-786 of SEQ ID NO:41,positions 691-872 of SEQ ID NO:92, SEQ ID NO:99, positions 567 to 718 ofSEQ ID NO:101, positions 483 to 634 of SEQ ID NO:102, positions 2-183 ofSEQ ID NO:105, positions 184-395 of SEQ ID NO:105, positions 396-578 ofSEQ ID NO:105, positions 579-761 of SEQ ID NO:105, positions 2-183 ofSEQ ID NO:106, 338-520 of SEQ ID NO:106, positions 478-629 of SEQ IDNO:107, positions 478-629 of SEQ ID NO:108, positions 478-629 of SEQ IDNO:109, positions 478-629 of SEQ ID NO:110, positions 400-581 of SEQ IDNO:112, positions 400-581 of SEQ ID NO:114, positions 400-581 of SEQ IDNO:116, positions 400-581 of SEQ ID NO:118, positions 400 to 581 of SEQID NO:120, positions 400 to 581 of SEQ ID NO:122, positions 400 to 581of SEQ ID NO:124, positions 400 to 581 of SEQ ID NO:126, positions 630to 811 of SEQ ID NO:128, positions 462 to 643 of SEQ ID NO:130,positions 688 to 869 of SEQ ID NO:132, positions 688 to 869 of SEQ IDNO:134, or a corresponding sequence from a different HBV strain. In oneaspect, the HBV antigens have an amino acid sequence that is at least95% identical, or at least 96% identical, or at least 97% identical, orat least 98% identical, or at least 99% identical, or is identical, toan amino acid sequence of SEQ ID NO:105, or a corresponding sequencefrom a different HBV strain.

Yet another embodiment of the invention relates to an immunotherapeuticcomposition comprising: (a) a yeast vehicle; and (b) a fusion proteincomprising at least two HBV Core proteins and at least two HBV Xantigens, where each of the HBV Core proteins is from a different HBVgenotype and where each of the HBV X antigens is from a different HBVgenotype. The composition elicits an HBV-specific immune response. Inone aspect, the HBV genotypes include genotype C; in one aspect, the HBVgenotypes include genotype D; in one aspect, the HBV genotypes includegenotype A; and in one aspect, the HBV genotypes include genotype B. Inone aspect, each of the HBV core proteins consists of at least 95% of afull-length HBV Core protein. In one aspect, each of the HBV coreproteins comprises amino acids 31 to 212 of HBV Core protein. In oneaspect, each of the HBV core proteins comprises amino acids 37 to 188 ofHBV Core protein. In one aspect, each of the HBV X antigens comprises atleast 95% of a full-length of HBV X antigen. In one aspect, each of theHBV X antigens comprises amino acids 52 to 127 of HBV X antigen.

In one aspect, the amino acid sequence of the HBV core antigen is atleast 95% identical, or at least 96% identical, or at least 97%identical, or at least 98% identical, or at least 99% identical, or isidentical, to an amino acid sequence selected from: positions 90-271 ofSEQ ID NO:95, positions 31-212 of SEQ ID NO:1, positions 31-212 of SEQID NO:5, positions 31-212 of SEQ ID NO:9, positions 37 to 188 of SEQ IDNO:9, positions 31-212 of SEQ ID NO:13, positions 31-212 of SEQ IDNO:17, positions 31-212 of SEQ ID NO:21, positions 14-194 of SEQ IDNO:25, positions 31-212 of SEQ ID NO:29, positions 408-589 of SEQ IDNO:34, positions 605 to 786 of SEQ ID NO:36, positions 352-533 of SEQ IDNO:38, positions 160-341 of SEQ ID NO:39, positions 605-786 of SEQ IDNO:41, positions 691-872 of SEQ ID NO:92, SEQ ID NO:99, positions 567 to718 of SEQ ID NO:101, positions 483 to 634 of SEQ ID NO:102, positions2-183 of SEQ ID NO:105, positions 184-395 of SEQ ID NO:105, positions396-578 of SEQ ID NO:105, positions 579-761 of SEQ ID NO:105, positions2-183 of SEQ ID NO:106, 338-520 of SEQ ID NO:106, positions 478-629 ofSEQ ID NO:107, positions 478-629 of SEQ ID NO:108, positions 478-629 ofSEQ ID NO:109, positions 478-629 of SEQ ID NO:110, positions 400-581 ofSEQ ID NO:112, positions 400-581 of SEQ ID NO:114, positions 400-581 ofSEQ ID NO:116, positions 400-581 of SEQ ID NO:118, positions 400 to 581of SEQ ID NO:120, positions 400 to 581 of SEQ ID NO:122, positions 400to 581 of SEQ ID NO:124, positions 400 to 581 of SEQ ID NO:126,positions 630 to 811 of SEQ ID NO:128, positions 462 to 643 of SEQ IDNO:130, positions 688 to 869 of SEQ ID NO:132, positions 688 to 869 ofSEQ ID NO:134, or a corresponding sequence from a different HBV strain.

In one aspect, the amino acid sequence of the HBV X antigen is at least95% identical, or at least 96% identical, or at least 97% identical, orat least 98% identical, or at least 99% identical, or is identical, toan amino acid sequence selected from: positions 90-242 of SEQ ID NO:96,SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, positions 2 to 154 of SEQ IDNO:12, SEQ ID NO:16, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:28, SEQ IDNO:32, positions 52-68 followed by positions 84-126 of SEQ ID NO:4,positions 52-68 followed by positions 84-126 of SEQ ID NO:8, positions52-68 followed by positions 84-126 of SEQ ID NO:12, positions 52-68followed by positions 84-126 of SEQ ID NO:16, positions 52-68 followedby positions 84-126 of SEQ ID NO:20, positions 52-68 followed bypositions 84-126 of SEQ ID NO:24, positions 52-68 followed by positions84-126 of SEQ ID NO:28, positions 52-68 followed by positions 84-126 ofSEQ ID NO:32, positions 787 to 939 of SEQ ID NO:36, positions 7-159 ofSEQ ID NO:39, positions 873-1025 of SEQ ID NO:92, SEQ ID NO:100,positions 719-778 of SEQ ID NO:101, positions 635-694 of SEQ ID NO:102,positions 184-337 of SEQ ID NO:106, positions 521-674 of SEQ ID NO:106,positions 630-689 of SEQ ID NO:107, positions 630-689 of SEQ ID NO:108,positions 630-689 of SEQ ID NO:109, positions 630-689 of SEQ ID NO:110,positions 582-641 of SEQ ID NO:122, positions 810-869 of SEQ ID NO:124,positions 582-641 of SEQ ID NO:126, positions 1-60 of SEQ ID NO:130,positions 229 to 288 of SEQ ID NO:132, positions 1 to 60 of SEQ IDNO:134, or a corresponding sequence from a different HBV strain.

In one aspect of this embodiment of the invention, the fusion proteinhas an amino acid sequence that is at least 95% identical, or at least96% identical, or at least 97% identical, or at least 98% identical, orat least 99% identical, or is identical, to an amino acid sequence ofSEQ ID NO:106, or a corresponding sequence from a different HBV strain.

In any of the embodiments described herein, including above and below,related to a fusion protein, HBV antigens, or immunotherapeuticcomposition comprising such a fusion protein or HBV antigens, in onefurther embodiment, the fusion protein can be appended at its N-terminusto add an additional sequence. In one aspect, the N-terminal sequence isselected from an amino acid sequence that is 95% identical to SEQ IDNO:37, an amino acid sequence that is 95% identical to SEQ ID NO:89, oran amino acid sequence that is 95% identical to SEQ ID NO:90. In oneaspect, the N-terminal sequence is selected from SEQ ID NO:37, positions1 to 5 of SEQ ID NO:37, SEQ ID NO:89, or SEQ ID NO:90, or acorresponding sequence from a different HBV strain.

In one aspect of any of the embodiments of the invention described aboveor elsewhere herein, the fusion protein is expressed by the yeastvehicle. In another aspect of any of the embodiments of the inventiondescribed above or elsewhere herein, the yeast vehicle is a whole yeast.The whole yeast, in one aspect is killed. In one aspect, the whole yeastis heat-inactivated.

In one aspect of any of any of the embodiments of the inventiondescribed above or elsewhere herein, the yeast vehicle can be from ayeast genus selected from: Saccharomyces, Candida, Cryptococcus,Hansenula, Kluyveromyces, Pichia, Rhodotorula, Schizosaccharomyces andYarrowia. In one aspect, the yeast vehicle is from Saccharomyces. In oneaspect, the yeast vehicle is from Saccharomyces cerevisiae.

In one aspect of any of the embodiments of the invention described aboveor elsewhere herein, the composition is formulated for administration toa subject or patient. In one aspect, the composition is formulated foradministration by injection of a subject or patient (e.g., by aparenteral route, such as subcutaneous or intraperitoneal orintramuscular injection). In one aspect, the composition is formulatedin a pharmaceutically acceptable excipient that is suitable foradministration to a human. In one aspect, the composition containsgreater than 90% yeast protein. In one aspect, the composition containsgreater than 90% yeast protein and is formulated for administration to apatient.

In one aspect of any of the embodiments of the invention described aboveor elsewhere herein, the fusion protein is not aggregated in the yeast.In one aspect, the fusion protein does not form inclusion bodies in theyeast. In one aspect, the fusion protein does not form VLPs or otherlarge antigen particles in the yeast. In one aspect, the fusion proteindoes form VLPs or other large antigen particles in the yeast.

In one aspect of any embodiment of the invention described above orelsewhere herein, in one aspect, the HBV sequences are from HBV genotypeA. In another aspect, the HBV sequences are from HBV genotype B. Inanother aspect, the HBV sequences are from HBV genotype C. In anotheraspect, the HBV sequences are from HBV genotype D. In another aspect,the HBV sequences are from HBV genotype E. In another aspect, the HBVsequences are from HBV genotype F. In another aspect, the HBV sequencesare from HBV genotype G. In another aspect, the HBV sequences are fromHBV genotype H. In one aspect, the HBV sequences are from a combinationof any of the above-referenced HBV genotypes or of any known HBVgenotypes or sub-genotypes.

Another embodiment of the invention relates to any of the fusionproteins described above as part of an immunotherapeutic composition ofthe invention, or elsewhere herein. In one aspect of this embodiment, afusion protein comprises HBV antigens, the HBV antigens selected from,but not limited to: (a) HBV antigens consisting of: HBV large surfaceantigen (L), HBV core protein and HBV X antigen; (b) HBV antigensconsisting of: HBV large surface antigen (L) and HBV core protein; (c)HBV antigens consisting of: hepatocyte receptor of Pre-S1 of the HBVlarge surface antigen (L), HBV small surface antigen (S), the reversetranscriptase domain of HBV polymerase, HBV core protein or HBVe-antigen, and HBV X antigen; (d) HBV antigens consisting of: HBV largesurface antigen (L), the reverse transcriptase domain of HBV polymerase,HBV core protein or HBV e-antigen, and HBV X antigen; (e) HBV antigensconsisting of: HBV large surface antigen (L), the reverse transcriptasedomain of HBV polymerase, and HBV core protein; (f) HBV antigensconsisting of: HBV polymerase (RT domain) and HBV core protein; (g) HBVantigens consisting of: HBV X antigen and HBV core protein; (h) HBVantigens consisting of: hepatocyte receptor of Pre-S1 of the HBV largesurface antigen (L), HBV small surface antigen (S), the reversetranscriptase domain of HBV polymerase, and HBV core protein or HBVe-antigen; (i) HBV antigens consisting of HBV large surface antigen (L);(j) HBV antigens consisting of HBV core antigen; (k) HBV antigensconsisting of: HBV polymerase including the reverse transcriptasedomain; (l) HBV antigens consisting of HBV X antigen; (m) HBV antigensconsisting of between two and four HBV surface antigens, HBV polymeraseantigens, HBV core antigens, or HBV X antigens, where each of thebetween two and four HBV antigens is from a different HBV genotype; and(n) HBV antigens consisting of two HBV core antigens and two HBV Xantigens, wherein each of the two HBV core antigens and each of the twoHBV X antigens are from a different HBV genotype. Aspects of theinvention related to each of the HBV antigens, including a variety ofsequences useful in these antigens, have been described above.

In one aspect of this embodiment of the invention, the fusion proteincomprises an amino acid sequence that is at least 95% identical, or atleast 96% identical, or at least 97% identical, or at least 98%identical, or at least 99% identical, or is identical, to an amino acidsequence selected from: SEQ ID NO:130, SEQ ID NO:150, SEQ ID NO:118, SEQID NO:151, SEQ ID NO:34, SEQ ID NO:120, SEQ ID NO:122, SEQ ID NO:124,SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:132, SEQ ID NO:134, SEQ IDNO:112, SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO:36, SEQ ID NO:38, SEQ IDNO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:92, SEQ ID NO:93, SEQ IDNO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ IDNO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:105, SEQID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, and SEQ IDNO:110.

Another embodiment of the invention relates to a recombinant nucleicacid molecule encoding any of the fusion proteins described herein. Inone aspect, the recombinant nucleic acid molecule comprises a nucleicacid sequence selected from, but not limited to: SEQ ID NO:33, SEQ IDNO:35, SEQ ID NO:91, SEQ ID NO:111, SEQ ID NO:113, SEQ ID NO:115, SEQ IDNO:117, SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:125, SEQID NO:127, SEQ ID NO:129, SEQ ID NO:131, or SEQ ID NO:133.

Yet another embodiment of the invention relates to an isolated celltransfected with any of the recombinant nucleic acid molecules describedherein. In one aspect, the cell is a yeast cell.

Another embodiment of the invention relates to a composition comprisingany of the fusion proteins described herein. Yet another embodiment ofthe invention relates to a composition comprising any of the recombinantnucleic acid molecules described herein. Another embodiment of theinvention relates to a composition comprising any of the isolated cellsdescribed herein.

Yet another embodiment of the invention relates to a method to treathepatitis B virus (HBV) infection or at least one symptom resulting fromHBV infection in a subject, comprising administering to a subject thatis infected with HBV at least one of any of the immunotherapeuticcompositions, including any HBV antigen, fusion protein, or yeast-basedimmunotherapeutic composition, described herein. The administration ofthe composition to the subject reduces HBV infection or at least onesymptom resulting from HBV infection in a subject.

Yet another embodiment of the invention relates to a method to elicit anantigen-specific, cell-mediated immune response against an HBV antigen,comprising administering to a subject any one or more of thecompositions, including any HBV antigen, fusion protein, or yeast-basedimmunotherapeutic composition, described herein.

Yet another embodiment of the invention relates to a method to preventHBV infection in a subject, comprising administering to a subject thathas not been infected with HBV, any one or more of the compositions,including any HBV antigen, fusion protein, or yeast-basedimmunotherapeutic composition, described herein.

Another embodiment of the invention relates to a method to immunize apopulation of individuals against HBV, comprising administering to thepopulation of individuals any one or more of the compositions, includingany HBV antigen, fusion protein, or yeast-based immunotherapeuticcomposition, described herein.

Another embodiment of the invention relates to any one or more of thecompositions, including any HBV antigen, fusion protein, or yeast-basedimmunotherapeutic composition, described herein, for use to treat HBVinfection or a symptom thereof.

Another embodiment of the invention relates to any one or more of thecompositions, including any HBV antigen, fusion protein, or yeast-basedimmunotherapeutic composition, described herein, for use to prevent HBVinfection or a symptom thereof.

Yet another embodiment of the invention relates to the use of any one ormore of the compositions, including any HBV antigen, fusion protein, oryeast-based immunotherapeutic composition, described herein in thepreparation of a medicament to treat HBV infection or a symptom thereof.

Yet another embodiment of the invention relates to the use of any one ormore of the compositions, including any HBV antigen, fusion protein, oryeast-based immunotherapeutic composition, described herein in thepreparation of a medicament to prevent HBV infection or a symptomthereof.

In one aspect of any of the embodiments related to methods or uses ofthe invention described above or elsewhere herein, the method caninclude administration of at least two, three, four or more of thecompositions, including any HBV antigen, fusion protein, or yeast-basedimmunotherapeutic composition, described herein. In one aspect,additional compositions or compounds useful for the prevention ortreatment of HBV infection can be administered (e.g., anti-viralcompounds, interferons, other immunotherapeutic compositions, orcombinations thereof). In one aspect, the various compositions orcompounds are administered concurrently to an individual. In one aspect,the various compositions or compounds are administered sequentially toan individual. In one aspect, each of the various compositions isadministered by injection to a different site on the individual. In oneaspect, a single dose of a yeast-based HBV immunotherapeutic compositionof the invention is between 40 Y.U. total and 80 Y.U. total,administered in equal parts at two, three or four different sites on anindividual, per dose.

In one aspect of any of the embodiments related to methods or uses ofthe invention described above or elsewhere herein, administration of thecomposition to the subject causes seroconversion in the subject orimproves seroconversion rates in a population of subjects. In oneaspect, administration of the composition to the subject reduces serumHBsAg or results in loss of serum HBsAg in the subject or improves ratesof loss of serum HBsAg in a population of subjects. In one aspect,administration of the composition to the subject reduces serum HBeAg orresults in loss of serum HBeAg in the subject or improves rates of lossof serum HBeAg in a population of subjects. In one aspect,administration of the composition to the subject reduces HBV viral loadin the subject or improves rates in reduction of HBV viral load in apopulation of subjects. In one aspect, administration of the compositionto the subject results in undetectable HBV DNA in infected cells in thesubject or results in higher rates of HBV DNA negativity in a populationof subjects. In one aspect, administration of the composition to thesubject reduces liver damage or improves liver function in the subjector reduces the rate of liver damage or increases the rate of improvedliver function in a population of subjects. In one aspect,administration of the composition to the subject improves ALTnormalization in the subject or in a population of subjects.

In any of the embodiments related to an HBV antigen, fusion protein,immunotherapeutic composition, or any method of use of the HBV antigen,fusion protein or immunotherapeutic composition described herein, in oneaspect, the composition further comprises, or is used in conjunctionwith, at least one biological response modifier. In one aspect, thecomposition further comprises, or is used in conjunction with, one ormore additional compounds useful for treating or ameliorating a symptomof HBV infection. In one aspect, the composition further comprises, oris used in conjunction with, at least one anti-viral compound. In oneaspect, the anti-viral is a nucleotide analogue reverse transcriptaseinhibitor. An anti-viral compound can include, but is not limited to,tenofovir, lamivudine, adefovir, telbivudine, entecavir, andcombinations thereof. In one aspect, the anti-viral compound istenofovir. In one aspect, the anti-viral compound is entecavir. In oneaspect, the composition further comprises, or is used in conjunctionwith, at least one interferon. In one aspect, the interferon isinterferon-α. In one aspect, the interferon is pegylated interferon-α2a.In one aspect, the interferon is interferon-λ.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic drawing showing the hepatitis B virus genomearrangement.

FIG. 2 is a schematic drawing showing the basic structure of arecombinant nucleic acid molecule encoding an HBV surface antigen/corefusion protein useful in a yeast-based immunotherapeutic composition ofthe invention.

FIG. 3 is a schematic drawing showing the basic structure of arecombinant nucleic acid molecule encoding an HBV surfaceantigen/polymerase/core/X fusion protein useful in a yeast-basedimmunotherapeutic composition of the invention.

FIG. 4 is a schematic drawing showing the basic structure of arecombinant nucleic acid molecule encoding an HBV polymerase/core fusionprotein useful in a yeast-based immunotherapeutic composition of theinvention.

FIG. 5 is a schematic drawing showing the basic structure of arecombinant nucleic acid molecule encoding an HBV X/core fusion proteinuseful in a yeast-based immunotherapeutic composition of the invention.

FIG. 6 is a schematic drawing showing the basic structure of arecombinant nucleic acid molecule encoding an HBV polymerase fusionprotein useful in a yeast-based immunotherapeutic composition of theinvention.

FIG. 7 is a schematic drawing showing the basic structure of arecombinant nucleic acid molecule encoding an HBV surfaceantigen/polymerase/core fusion protein useful in a yeast-basedimmunotherapeutic composition of the invention.

FIG. 8 is a schematic drawing showing the basic structure of arecombinant nucleic acid molecule encoding an HBV surfaceantigen/core/polymerase fusion protein useful in a yeast-basedimmunotherapeutic composition of the invention.

FIG. 9 is a schematic drawing showing the basic structure of arecombinant nucleic acid molecule encoding an HBV surface antigen/core/Xfusion protein useful in a yeast-based immunotherapeutic composition ofthe invention.

FIG. 10 is a schematic drawing showing the basic structure of arecombinant nucleic acid molecule encoding an HBV surfaceantigen/core/polymerase/X fusion protein useful in a yeast-basedimmunotherapeutic composition of the invention.

FIG. 11 is a schematic drawing showing the basic structure of arecombinant nucleic acid molecule encoding an HBV surfaceantigen/core/X/polymerase fusion protein useful in a yeast-basedimmunotherapeutic composition of the invention.

FIG. 12 is a schematic drawing showing the basic structure of arecombinant nucleic acid molecule encoding an HBV polymerase/surfaceantigen/core fusion protein useful in a yeast-based immunotherapeuticcomposition of the invention.

FIG. 13 is a schematic drawing showing the basic structure of arecombinant nucleic acid molecule encoding an HBV X/surface antigen/corefusion protein useful in a yeast-based immunotherapeutic composition ofthe invention.

FIG. 14 is a schematic drawing showing the basic structure of arecombinant nucleic acid molecule encoding an HBV polymerase/X/surfaceantigen/core fusion protein useful in a yeast-based immunotherapeuticcomposition of the invention.

FIG. 15 is a schematic drawing showing the basic structure of arecombinant nucleic acid molecule encoding an HBV X/polymerase/surfaceantigen/core fusion protein useful in a yeast-based immunotherapeuticcomposition of the invention.

FIG. 16 is a digital image of a Western blot showing expression ofseveral yeast-based immunotherapeutic compositions expressing an HBVSurface antigen/Core fusion protein (heat-killed, whole yeast).

FIG. 17 is a digital image of a Western blot showing expression ofseveral yeast-based immunotherapeutic compositions expressing an HBVSurface antigen/Core fusion protein (live, whole yeast).

FIG. 18 is a digital image of a Western blot showing expression ofseveral yeast-based immunotherapeutic compositions expressing an HBVsurface antigen/polymerase/core/X fusion protein.

FIG. 19 is a digital image of a Western blot showing expression ofseveral yeast-based immunotherapeutic compositions expressing an HBVsurface antigen/polymerase/core/X fusion protein.

FIG. 20 is a digital image of a Western blot showing expression ofseveral yeast-based immunotherapeutic compositions expressing HBVantigens comprising surface-core fusion proteins (Sc) orsurface-polymerase-core-X fusion proteins (Sp).

FIG. 21 is a digital image of a Western blot showing expression of HBVantigens from several yeast-based HBV immunotherapeutic compositionscultured in UL2 medium.

FIG. 22 is a bar graph showing the average expression of HBV antigensfrom several yeast-based HBV immunotherapeutic compositions cultured inUL2 medium or U2 medium (error bars are Standard Deviation).

FIG. 23 is a graph showing the proliferation of splenic CD4⁺ T cellsfrom mice immunized with a yeast-based immunotherapeutic productexpressing an HBV Surface-Core antigen (SCORE) to an S/Core antigen mixor to a MHC Class II SAg mimetope peptide (error bars are StandardDeviation).

FIG. 24 is a graph showing the proliferation of lymph node T cells frommice immunized with a yeast-based immunotherapeutic product expressingan HBV Surface-Core antigen (SCORE) to an S/Core antigen mix or to a MHCClass II SAg mimetope peptide (error bars are Standard Deviation).

FIG. 25 is a graph showing the interferon-γ (IFN-γ) ELISpot response oflymph node T cells from mice immunized with a yeast-basedimmunotherapeutic product expressing an HBV Surface-Core antigen (SCORE)to an S/Core antigen mix or to a MHC Class II SAg mimetope peptide.

FIG. 26 is a graph showing the proliferation of splenic CD4⁺ T cellsfrom mice immunized with a yeast-based immunotherapeutic productexpressing an HBV Surface-Pol-Core-X antigen (denoted a-Spex) to anS/Core antigen mix or to a MHC Class II SAg mimetope peptide (error barsare Standard Deviation).

FIG. 27 is a graph showing IL-1β production in splenocytes from miceimmunized with: (a) a yeast-based immunotherapeutic product expressingan HBV Surface-Pol-E/Core-X antigen (denoted Sp), left columns; or (b) ayeast-based immunotherapeutic product expressing an HBV Surface-Coreantigen (denoted Sc) (error bars are Standard Deviation).

FIG. 28 is a graph showing IL-12p70 production in splenocytes from miceimmunized with: (a) a yeast-based immunotherapeutic product expressingan HBV Surface-Pol-Core-X antigen (denoted Sp), left columns; or (b) ayeast-based immunotherapeutic product expressing an HBV Surface-Coreantigen (denoted Sc) (error bars are Standard Deviation).

FIGS. 29A and 29B are graphs showing interferon-γ (IFN-γ) production insplenocytes from mice immunized with: (FIG. 29A) a yeast-basedimmunotherapeutic product expressing an HBV Surface-Core antigen(denoted Sc) or (FIG. 29B) a yeast-based immunotherapeutic productexpressing an HBV Surface-Pol-Core-X antigen (denoted Sp) (error barsare Standard Deviation).

FIGS. 30A-D are graphs showing IL-1β (FIG. 30A), IL-6 (FIG. 30B), IL-13(FIG. 30C), and IL-12p70 (FIG. 30D) production in splenocytes from miceimmunized with: (a) a yeast-based immunotherapeutic product expressingan HBV Surface-Pol-Core-X antigen (denoted Sp), left columns; or (b) ayeast-based immunotherapeutic product expressing an HBV Surface-Coreantigen (denoted Sc).

FIG. 31 is a bar graph showing that mice immunized with GI-13002 orGI-13002+anti-CD40 antibody, but not YVEC, elicited comparableprotection from challenge with EL4 tumors expressing the target HBVantigen (error bars are Standard Deviation).

FIG. 32 is a bar graph showing the results of an IFN-γ ELISpot assaycomparing T cell responses of mice immunized with GI-13008 (SCORE-C) andGI-13013 (SPEXv2) as compared to YVEC using a variety of HBV peptidesand antigens (error bars are Standard Deviation).

FIG. 33 is a bar graph showing IFN-γ ELISpot responses to stimulationwith GI-13002 from a human subject pre- and post-immunization, andpost-boost, with a prophylactic HBV vaccine.

FIG. 34 is a bar graph showing HBV antigen-specific IFN-γ ELISpotresponses from lymph node cells isolated from HLA-A2 transgenic miceimmunized with GI-13009 (SCORE-D) or GI-13020 (X-SCORE) as compared tomice immunized with a yeast control (YVEC) (error bars are StandardDeviation).

FIG. 35 is a bar graph showing HBV antigen-specific IFN-γ ELISpotresponses from spleen cells isolated from HLA-A2 transgenic miceimmunized with GI-13009 (SCORE-D) as compared to mice immunized with ayeast control (YVEC) (error bars are Standard Error).

FIG. 36 is a bar graph showing HBV antigen-specific IFN-γ ELISpotresponses from lymph node cells isolated from C57BL/6 mice immunizedwith GI-13009 (SCORE-D) or GI-13020 (X-SCORE) as compared to miceimmunized with a yeast control (YVEC) or Naïve mice (error bars areStandard Deviation).

FIG. 37 is a line graph showing HBV antigen-specific CD8⁺ T cellresponses to an MHC Class I-restricted HBV peptide in C57BL/6 miceimmunized with GI-13009 (SCORE-D) or GI-13020 (X-SCORE) as compared tomice immunized with a yeast control (YVEC) or a yeast-basedimmunotherapeutic expressing ovalbumin (OVAX).

FIG. 38 is a bar graph showing HBV antigen-specific CD4⁺ T cellresponses to an MHC Class II-restricted HBV peptide in C57BL/6 miceimmunized with GI-13009 (SCORE-D) or GI-13020 (X-SCORE) as compared tomice immunized with a yeast control (YVEC) or a yeast-basedimmunotherapeutic expressing ovalbumin (OVAX).

DETAILED DESCRIPTION OF THE INVENTION

This invention generally relates to compositions and methods forpreventing and/or treating hepatitis B virus (HBV) infection. Theinvention includes a yeast-based immunotherapeutic composition (alsoreferred to as “yeast-based HBV immunotherapy”) comprising a yeastvehicle and HBV antigen(s) that have been designed to elicit aprophylactic and/or therapeutic immune response against HBV infection inan individual, and the use of such compositions to prevent and/or treatHBV infection and related symptoms thereof. The invention also includesthe recombinant nucleic acid molecules used in the yeast-basedcompositions of the invention, as well as the proteins and fusionproteins encoded thereby, for use in any immunotherapeutic compositionand/or any therapeutic or prophylactic protocol for HBV infection,including any therapeutic or prophylactic protocol that combines theHBV-specific yeast-based compositions of the invention with any one ormore other therapeutic or prophylactic compositions, agents, drugs,compounds, and/or protocols for HBV infection.

The yeast-based, HBV-specific immunotherapeutic compositions are uniqueamong various types of immunotherapy, in that these compositions of theinvention induce innate immune responses, as well as adaptive immuneresponses that specifically target HBV, including CD4-dependent TH17 andTH1 T cell responses and antigen-specific CD8⁺ T cell responses. Thebreadth of the immune response elicited by HBV-specific yeast-basedimmunotherapy is well-suited to target HBV. First, HBV is believed toevade the innate immune response early in infection by “hiding” from theinnate response and thereby not inducing it, rather than by directlycounteracting innate immunity (Wieland and Chisari, 2005, J. Virol.15:9369-9380; Wieland, et al., 2004, PNAS USA 101:6669-6674).Accordingly, it can be expected that HBV will be sensitive to innateimmune responses if they are activated by another mechanism, i.e., theyeast-based immunotherapeutic compositions of the invention. Second, HBVproduces high-level antigen expression in infected host cells that isexpected to be visible to the adaptive immune response (Guidotti, etal., 1999, Science 284:825-829; Thimme et al., 2003, J. Virol.77:68-76), and clearance of acute infection has been associated withrobust CD4⁺ and CD8⁺ T cell responses (Maini et al., 1999,Gastroenterol. 117:1386-1396; Rehermann et al., 1995, J. Exp. Med.181:1047-1058; Thimme et al., 2003, J. Virol. 77:68-76; Wieland andChisari, 2005, J. Virol. 15:9369-9380). Therefore, yeast-based HBVimmunotherapy, by activating the adaptive immune response, is expectedto effectively target HBV-infected cells for destruction and/or isexpected to effectively enhance viral clearance. Moreover, the immuneresponse generated by yeast-based immunotherapy is believed to beinterferon-independent and interferon-dependent (Tamburini et al., 2012,J. Immunother. 35(1):14-22); accordingly, the ability, or lack thereof,of an individual to respond to interferon-based therapy, which is onestandard of care treatment for HBV, is not believed to directly impactthe ability of the subject to respond to yeast-based immunotherapy ofthe invention. In addition, the yeast-based HBV immunotherapycompositions described herein are designed to target immunogenic andconserved regions of HBV, multiple CTL epitopes, and include regions ofHBV that may be targeted for escape (allowing for modifications of thecompositions as needed to target such escape mutations), making it ahighly adaptable therapy for HBV that optimizes the opportunity foreffective immune responses against this virus.

In addition, and without being bound by theory, yeast-basedimmunotherapy for HBV is believed to induce an immune response that isnot only directed specifically against the target antigen carried by theyeast-based immunotherapeutic product, but that also evolves to bedirected against other immunological epitopes on the virus (i.e., otherthan those carried by the yeast-antigen composition). In other words, aprimary cellular immune response to the antigen(s) and/or epitope(s)contained in the yeast-based immunotherapeutic can lead to secondarycellular immune responses to antigen(s) and/or epitope(s) that arepresent in the infected cells in the treated subject but that are notpresent in the yeast-based immunotherapeutic, thereby leading to theevolution of complex and unpredictable immune response profiles that areunique to each treated subject. These secondary immune responses arespecific to the molecular profile of the HBV infection in each subjecttreated, and the yeast-based immunotherapeutic may drive thesedownstream effects in a unique manner when compared to other treatmentmodalities, including other immunotherapy platforms. This phenomenon mayalso be generally referred to as “epitope spreading” and represents anadvantage of using yeast-based HBV immunotherapy, because induction ofan immune response against a particular HBV antigen or even against aparticular HBV genotype (e.g., by providing that antigen in the contextof the yeast immunotherapeutic), is expected to result in the cascadingtargeting of the immune system against a variety of additional HBVantigens, which may result in effective immune responses againstantigens from different HBV genotypes or strains than those representedin the yeast-based immunotherapeutic composition.

As discussed above, patients who become chronically infected with HBVtend to have weaker (or absent) and more narrow HBV-specific, Tcell-mediated immunity. Accordingly, the yeast-based HBV immunotherapycompositions of the invention address the need for therapeuticcompositions to treat patients who are actively infected with HBV,including chronically infected patients, and further provide anadditional vaccine for the prevention of HBV infection that may haveadvantages with respect to the production of durable memory immuneresponses. Indeed, the yeast-based HBV immunotherapy compositions of theinvention are expected to promote durable memory T cell responsesagainst HBV, which can prevent infection, as well as provide long termbenefits that can protect a chronically infected patient from viralreactivation. Yeast-based HBV immunotherapy compositions as monotherapyor in combination with other therapeutic approaches for the treatment ofHBV (e.g., in combination with anti-viral compounds) are expected toincrease the percentage of chronically infected patients who achieveclearance of HBsAg and HBeAg, who achieve complete seroconversion,and/or who achieve sustained viral clearance for at least 6 months afterthe completion of therapy.

Accordingly, yeast-based HBV immunotherapy can be combined withanti-viral drugs and/or interferon therapy, and/or with other therapiesfor HBV, in order to reduce the viral load in an individual to a levelthat can be more effectively handled by the immune system. HBV viraltiters are typically very high (as many as 10¹¹ hepatocytes may beinfected) and thus may overwhelm an individual's ability to mount aneffective CTL response; accordingly, reduction of viral load usinganti-viral drugs in combination with induction of HBV-specific CTLactivity using yeast-based immunotherapy is expected to be beneficial tothe infected individual. In addition, reduction of viral load throughthe use of anti-viral drugs may also reduce negative effects, if any, ofimmune activation in the context of a high number of infectedhepatocytes being targeted for destruction. Yeast-based HBVimmunotherapy is also expected to play a role in reducing and/oreliminating compartments of latent viral infection. For example, thereare many tissues that have been shown to be HBV-positive by PCR, andthat are considered potential sanctuaries for re-activation of HBV. HBVDNA can integrate into the host genome, which provides for a quiescentpersistence of HBV, and cccDNA is a supercoiled, dormant form of the HBVgenome that also contributes to quiescence. Without being bound bytheory, the inventors believe that yeast-based HBV immunotherapydescribed herein will play a role in eliminating all of these types ofHBV “sanctuaries” that likely contribute to the low disease-free curerate observed with the current anti-viral approaches.

In another scenario, use of a yeast-based HBV immunotherapeutic of theinvention, alone or in combination with an anti-viral or other HBVtherapeutic, if sufficient to achieve complete clearance of HBsAg, butnot sufficient to achieve anti-HB production, may be followed by, orfurther combined with, existing prophylactic subunit vaccines to achievecomplete seroconversion. Alternatively, any of the fusion proteinsdescribed herein may also be used as subunit vaccines to achievecomplete seroconversion, or to protect a subject from HBV infection,alone or in combination with a yeast-based HBV immunotherapeutic of theinvention. Finally, the immunotherapeutic composition of the inventionis well-suited for modification and/or combination with additionalimmunotherapeutic compositions, including any described herein, to treatescape mutations of HBV that are elicited by treatment with anti-viraldrugs.

Yeast-based immunotherapeutic compositions are administered as biologicsor pharmaceutically acceptable compositions. Accordingly, rather thanusing yeast as an antigen production system followed by purification ofthe antigen from the yeast, the entire yeast vehicle as described hereinmust be suitable for, and formulated for, administration to a patient.In contrast, existing commercial HBV vaccines as well as many indevelopment, comprise recombinant HBV proteins (e.g., HBsAg proteins)that are produced in Saccharomyces cerevisiae, but are subsequentlyreleased from the yeast by disruption and purified from the yeast sothat the final vaccine, combined with an adjuvant (e.g., aluminumhydroxyphosphate sulfate or aluminum hydroxide), contains no detectableyeast DNA and contains no more than 1-5% yeast protein. The HBVyeast-based immunotherapeutic compositions of the invention, on theother hand, contain readily detectable yeast DNA and containsubstantially more than 5% yeast protein; generally, yeast-basedimmunotherapeutics of the invention contain more than 70%, more than80%, or generally more than 90% yeast protein.

Yeast-based immunotherapeutic compositions are administered to a patientin order to immunize the patient for therapeutic and/or prophylacticpurposes. In one embodiment of the invention, the yeast-basedcompositions are formulated for administration in a pharmaceuticallyacceptable excipient or formulation. The composition should beformulated, in one aspect, to be suitable for administration to a humansubject (e.g., the manufacturing conditions should be suitable for usein humans, and any excipients or formulations used to finish thecomposition and/or prepare the dose of the immunotherapeutic foradministration should be suitable for use in humans). In one aspect ofthe invention, yeast-based immunotherapeutic compositions are formulatedfor administration by injection of the patient or subject, such as by aparenteral route (e.g., by subcutaneous, intraperitoneal, intramuscularor intradermal injection, or another suitable parenteral route).

In one embodiment, the yeast express the antigen (e.g., detectable by aWestern blot), and the antigen is not aggregated in the yeast, theantigen does not form inclusion bodies in the yeast, and/or does notform very large particles (VLPs) or other large antigen particles in theyeast. In one embodiment, the antigen is produced as a soluble proteinin the yeast, and/or is not secreted from the yeast or is notsubstantially or primarily secreted from the yeast. In anotherembodiment, without being bound by theory, the present inventors believethat particular combinations and perhaps, arrangements, of antigens inan HBV fusion protein including surface antigen and core antigen,described in detail herein, may form VLPs or aggregate to some extentwithin the yeast expressing the antigens. As a result, the antigenexpressed by the yeast has immunogenic properties which appear to berelated to its overall structure and form, as a separate characteristicfrom the immunogenic properties of the immune epitopes (e.g., T cellepitopes) carried within the antigen. When the yeast expressing suchfusion proteins are provided in a yeast-based HBV immunotherapeutic ofthe invention, the immunotherapeutic composition derives properties thatactivate the innate immune system not only from the yeast vehicle asdiscussed above (as with all yeast-based immunotherapeutics describedherein), but also in part from the fusion protein antigen structure(e.g., the surface-core fusion protein as expressed in the yeast alsohas adjuvant-like properties); in addition, the immunotherapeuticcomposition derives properties that activate the adaptive immune systemin an antigen-specific manner from the fusion protein (via provision ofvarious T cell epitopes), as with all of the yeast-basedimmunotherapeutics described herein. This specific combination ofproperties appears to be unique to yeast-based immunotherapeuticsexpressing particular surface-core fusion proteins from HBV describedherein. However, in all of the embodiments of the invention describedherein, the yeast-based immunotherapeutics should be readilyphagocytosed by dendritic cells of the immune system, and the yeast andantigens readily processed by such dendritic cells, in order to elicitan effective immune response against HBV.

Compositions of the Invention

One embodiment of the present invention relates to a yeast-basedimmunotherapy composition which can be used to prevent and/or treat HBVinfection and/or to alleviate at least one symptom resulting from theHBV infection. The composition comprises: (a) a yeast vehicle; and (b)one or more antigens comprising HBV protein(s) and/or immunogenicdomain(s) thereof. In conjunction with the yeast vehicle, the HBVproteins are most typically expressed as recombinant proteins by theyeast vehicle (e.g., by an intact yeast or yeast spheroplast, which canoptionally be further processed to a yeast cytoplast, yeast ghost, oryeast membrane extract or fraction thereof), although it is anembodiment of the invention that one or more such HBV proteins areloaded into a yeast vehicle or otherwise complexed with, attached to,mixed with or administered with a yeast vehicle as described herein toform a composition of the present invention. According to the presentinvention, reference to a “heterologous” protein or “heterologous”antigen, including a heterologous fusion protein, in connection with ayeast vehicle of the invention, means that the protein or antigen is nota protein or antigen that is naturally expressed by the yeast, althougha fusion protein that includes heterologous antigen or heterologousprotein may also include yeast sequences or proteins or portions thereofthat are also naturally expressed by yeast (e.g., an alpha factor preprosequence as described herein).

One embodiment of the invention relates to various HBV fusion proteins.In one aspect, such HBV fusion proteins are useful in a yeast-basedimmunotherapeutic composition of the invention. Such fusion proteins,and/or the recombinant nucleic acid molecules encoding such proteins,can also be used in, in combination with, or to produce, anon-yeast-based immunotherapeutic composition, which may include,without limitation, a DNA vaccine, a protein subunit vaccine, arecombinant viral-based immunotherapeutic composition, a killed orinactivated pathogen vaccine, and/or a dendritic cell vaccine. Inanother embodiment, such fusion proteins can be used in a diagnosticassay for HBV and/or to generate antibodies against HBV. Describedherein are exemplary HBV fusion proteins providing selected portions ofHBV antigens, including, for example, selected portions of and/ormodified polymerase; selected portions of and/or modified surfaceantigen; selected portions of and/or modified core (including at leastportions of or most of e-antigen); selected portions of and/or modifiedX antigen; as well as selected portions of and/or arrangements of anyone, two, three or all four of the antigens (surface antigen, core, Xand polymerase), such as, but not limited to, selected portions and/orarrangements of surface antigen and core (including at least portions ofor most of e-antigen); selected portions and/or arrangements of surfaceantigen, core (including at least portions of or most of e-antigen),polymerase and X antigen; selected portions and/or arrangements ofsurface antigen, core (including at least portions of or most ofe-antigen), and polymerase; and selected portions and/or arrangements ofsurface antigen, core (including at least portions of or most ofe-antigen), and X antigen.

In one embodiment, HBV antigens, including immunogenic domains offull-length proteins, as described herein, are fused to host proteinsthat are overexpressed in HBV infected, but not in non-infected, hostcells. In one embodiment, HBV antigens, including immunogenic domains offull-length proteins, as described herein, are fused to protein R2, ahost factor required for HBV replication, which in one embodiment, isexpressed in hepatocytes. R2 is a protein component of ribonucleotidereductase (RNR), and is critical for the HBV life-cycle (see, e.g.,Cohen et al., 2010, Hepatol. 51(5):1538-1546). Other embodiments of theinvention will be apparent in view of the disclosure provided herein.

Hepatitis B Virus, Genes, and Proteins.

Hepatitis B virus (HBV) is a member of the Hepadnaviridae (hepadnavirus)family of viruses and causes transient and chronic infections of theliver in humans and the great apes. The hepadnaviruses that infectmammals have similar DNA sequences and genome organization, and aregrouped in the genus Orthohepadnavirus. The hepatitis B virus particlehas an outer envelope containing lipid and surface antigen particlesknown as HBsAg. A nucleocapsid core containing core protein (HBcAg)surrounds the viral DNA and a DNA polymerase with reverse transcriptaseactivity. As reviewed in Seeger and Mason, 2000, Microbiol. Mol. Biol.Rev. 64(1):51-68, HBV has a 3.2 kb partially double-strandedrelaxed-circular DNA (rcDNA) genome that is converted into a covalentlyclosed circular double-stranded DNA (cccDNA) molecule upon delivery ofthe viral genome to the nucleus of an infected hepatocyte. The host cellRNA polymerase II transcribes four viral RNAs from the cccDNA templatewhich are transported to the host cell cytoplasm. The viral RNAs includemRNAs that are transcribed to produce the viral core and envelopestructural proteins and the precore, polymerase and X nonstructuralviral proteins. The RNA that is translated to produce core andpolymerase also serves as the pregenomic RNA (pgRNA) which is thetemplate for reverse transcription. pgRNA and the polymerase areencapsulated by the core protein, producing the viral nucleocapsid wherethe pgRNA is reverse transcribed into rcDNA. These rcDNA-containingnucleocapsids are then enclosed by envelope proteins and secreted fromthe host cell as mature virions or shuttled to the nucleus to amplifythe viral cccDNA.

The structural and non-structural proteins produced by the HBV genomeare shown in Table 1. The partially double-stranded HBV genome containsfour genes known as C, X, P, and S (see also FIG. 1).

TABLE 1 HBV genes and gene products Gene Protein Function(s) C coreprotein (HBcAg) Forms viral capsid surrounding viral pgRNA andpolymerase e antigen (HBeAg) Function unknown; may be HBV-specificimmune suppressive factor for adaptive immune response P polymerasePolymerase for viral DNA replication Domain 1: terminal protein (TP)domain packages pgRNA and primes minus strand DNA Domain 2: reversetranscriptase (RT) domain, RNase H; degrades pgRNA S S HBsAg (surfaceEnvelope protein and forms antigen; small) surface antigen particles;may suppress immune function M HBsAg (surface Envelope protein and formsantigen; surface antigen particles middle = together with S; maysuppress Pre-S2 + S) immune function L HBsAg (surface Envelope proteinand antigen; large = forms surface antigen particles Pre-S1 + togetherwith S; pre-S1 domain pre-S2 + S) provides ligand for core particlesduring assembly of viral envelope; hepatocyte receptor; may suppressimmune function X X antigen (HBx) Transcriptional transactivation;regulation of DNA repair pathways; elevation of cytosolic calciumlevels; modulation of protein degradation pathways; modulation of cellcycle progression and cell proliferation pathways in host cell;stimulation of HBV replication

Gene C encodes two closely related antigens: a 21-kDa protein called“core protein” or “core antigen” (HBcAg) which forms the viral capsid,and a 17-kDa protein called e-antigen (HBeAg) that forms dimers but thatdoes not assemble into capsid. Full-length core protein is anapproximately 183 amino acid protein, comprising all but the N-terminal10 amino acids of e-antigen and comprising approximately 34 additionalamino acids at the C-terminus that are proteolytically cleaved in theproduction of e-antigen. In other words, core protein and e-antigen have149 amino acid residues in common (this section sometimes being referredto as the hepatitis core antigen), but differ at the N-terminal andC-terminal regions. Precore protein is a precursor protein comprising anamino acid sequence that includes sequence from both core and e-antigen,from which e-antigen is produced via proteolytic processing.Intracellular HBeAg includes precore residues −29 to −1 (the residuenumbering in this particular description is provided with the firstamino acid residue of core protein within the precore protein beingdenoted as position “1”), which contains a signal sequence that directsthe protein to the endoplasmic reticulum at which point amino acids −29to −11 are cleaved; another proteolytic cleavage between amino acids 149and 150 removes the C-terminal portion of precore (which is present infull-length core protein), and the remaining HBeAg (consisting of aminoacids −10 to −1 of precore plus amino acids 1-149 of HBcAg or core) isthen secreted as e-antigen (Standing et al., 1988, PNAS USA 85:8405-8409; Ou et al., 1986, PNAS USA 83:1578-1582; Bruss and Gerlich,1988, Virology 163:268-275; Takahashi et al., 1983, J. Immunol.130:2903-2907). HBeAg consisting of the entire precore region has alsobeen found in human sera (Takahashi et al., 1992, J. Immunol.147:3156-3160). As mentioned, HBcAg (core) forms dimers that assembleinto the viral capsid and contain the polymerase and viral DNA or pgRNA.The function of HBeAg (e-antigen) is unknown, but it is not required forHBV replication or infection, and it is thought to be an immunesuppressive factor that protects HBV against attack by the immune system(Milich et al., 1990, PNAS USA 87:6599-6603; Che et al., 2004, PNAS USA101:14913-14918; Wieland and Chisari, 2005, J. Virol. 79:9369-9380). Forclarity, in the HBV sequences described herein (e.g., see Table 3), thesequence for precore from representative HBV genotypes is provided, andthe positions of core protein and e-antigen are denoted within theprecore sequence, with the first amino acid of precore designated asposition 1.

Gene P encodes the HBV DNA polymerase (Pol), which consists of two majordomains linked by a spacer. The N-terminal domain of the polymerase(also referred to as “terminal protein” or TP) is involved in thepackaging of pgRNA and in the priming of non-sense strand DNA. TheC-terminal domain is a reverse transcriptase (RT) that has RNase H (RH)activity.

Gene S has multiple start codons and encodes three envelope proteins(also referred to herein generally as “surface protein” or “surfaceantigen”) denoted S, M and L, which are all components of the infectiousviral particles, also known as Dane particles. S, by itself, andtogether with M and L, also form surface antigen particles (HBsAg) whichcan be secreted from infected cells in large quantities (Seeger andMason, 2000, Microbiol. Mol. Biol. Rev. 64(1):51-68; Beck, (2007),“Hepatitis B virus replication”, World Journal of Gastroenterology: WJG13(1):48-64). The codons for M and L are located approximately 165 (M)and 489 (L) nucleotides, respectively, upstream of the initiation codonfor S. S or “small” surface antigen is the smallest and most abundant ofthe surface antigens. Antibodies produced against this antigen representseroconversion in infected individuals. M or “middle” surface antigenhas an extra protein domain, as compared to S, known as pre-S2, and theprotein domain that is unique to L or “large” surface antigen is knownas pre-S1 (L therefore also contains pre-S2 and the additional sequencebelonging to M and S). Pre-S1 contains the viral hepatocyte receptordomain (hepatocyte receptor binding site), which is locatedapproximately between amino acid positions 21 and 47 of Pre-S1. Epitopesin pre-S1 can elicit virus-neutralizing antibodies. In addition, thepre-S1 domain provides the ligand for core particles during the assemblyof the viral envelope. Surface antigen particles (HBsAg) may alsosuppress immune elimination of infected cells by functioning as ahigh-dose toleragen (Reignat et al., 2002, J. Exp. Med. 195:1089-1101;Webster et al., 2004, J. Virol. 78:5707-5719).

Gene X encodes X antigen (HBx) (which may also be referred to as “Xprotein”) which is involved in transcriptional transactivation,regulation of DNA repair pathways, elevation of cytosolic calciumlevels, modulation of protein degradation pathways, and modulation ofcell cycle progression and cell proliferation pathways in the host cell(Gearhart et al., 2010, J. Virol.), which enhances stimulation of HBVreplication. HBx is also associated with the development of liver cancer(Kim et al., Nature 1991, 351:317-320; Terradillos et al., Oncogene1997, 14:395-404).

HBV is found as one of four major serotypes (adr, adw, ayr, ayw) thatare determined based on antigenic epitopes within its envelope proteins.There are eight different HBV genotypes (A-H) based on the nucleotidesequence variations in the genome. The geographical distribution of thegenotypes is shown in Table 2 (Kramvis et al., 2005, Vaccine23(19):2409-2423; Magnius and Norder, 1995, Intervirology 38(1-2):24-34;Sakamoto et al., 2006, J Gen. Virol. 87:1873-1882; Lim et al., 2006,Int. J. Med. Sci. 3:14-20).

TABLE 2 HBV genotype Prevalent Geographical Distribution HBV/A Americas,Europe, Africa, Southeast Asia HBV/B Asia (China, Japan, SoutheastAsia), United States HBV/C Asia (China, Japan, Southeast Asia), UnitedStates HBV/D United States, Mediterranean, Middle East and India HBV/ESub-Saharan and West Africa HBV/F Central and South America HBV/GFrance, Germany, United States HBV/H Central America, United States(California)

The nucleic acid and amino acid sequence for HBV genes and the proteinsencoded thereby are known in the art for each of the known genotypes.Table 3 provides reference to sequence identifiers for exemplary(representative) amino acid sequences of all of the HBV structural andnon-structural proteins in each of the eight known genotypes of HBV, andfurther indicates the position of certain structural domains. It isnoted that small variations may occur in the amino acid sequence betweendifferent viral isolates of the same protein or domain from the same HBVgenotype. However, as discussed above, strains and serotypes of HBV andgenotypes of HBV display high amino acid identity even between serotypesand genotypes (e.g., see Table 4). Therefore, using the guidanceprovided herein and the reference to the exemplary HBV sequences, one ofskill in the art will readily be able to produce a variety of HBV-basedproteins, including fusion proteins, from any HBV strain (isolate),serotype, or genotype, for use in the compositions and methods of thepresent invention, and as such, the invention is not limited to thespecific sequences disclosed herein. Reference to an HBV protein or HBVantigen anywhere in this disclosure, or to any functional, structural,or immunogenic domain thereof, can accordingly be made by reference to aparticular sequence from one or more of the sequences presented in thisdisclosure, or by reference to the same, similar or correspondingsequence from a different HBV isolate (strain).

TABLE 3 Organism, Sequence Genotype, Identifier Gene Protein (DatabaseAccession No.) HBV, Precore SEQ ID NO: 1 Genotype A, (Accession No.AAX83988.1) C Core (HBcAg) *Positions 30/31-212 of SEQ ID NO: 1e-antigen (HBeAg) *Positions 20-178 of SEQ ID NO: 1 HBV, Polymerase SEQID NO: 2 Genotype A, (Accession No. BAI81985) P reverse transcriptase*Positions 383-602 of SEQ ID NO: 2 HBV, Surface HBsAg (L) SEQ ID NO: 3Genotype A, (Accession No. BAD91280.1) S Surface HBsAg (M) *Positions120-400 of SEQ ID NO: 3 Surface HBsAg (S) *Positions 175-400 of SEQ IDNO: 3 HBV, X (HBx) SEQ ID NO: 4 Genotype A, (Accession No. AAK97189.1) XHBV, Precore SEQ ID NO: 5 Genotype B, (Accession No. BAD90067) C Core(HBcAg) *Positions 30/31-212 of SEQ ID NO: 5 e-antigen (HBeAg)*Positions 20-178 of SEQ ID NO: 5 HBV, Polymerase SEQ ID NO:6 GenotypeB, (Accession No. BAD90068.1) P reverse transcriptase *Positions 381-600of SEQ ID NO: 6 HBV, Surface HBsAg (L) SEQ ID NO: 7 Genotype B, S(Accession No. BAJ06634.1) Surface HBsAg (M) *Positions 120-400 of SEQID NO: 7 Surface HBsAg (S) *Positions 175-400 of SEQ ID NO: 7 HBV, X(HBx) SEQ ID NO: 8 Genotype B, X (Accession No. BAD90066.1) HBV, PrecoreSEQ ID NO:9 Genotype C, C (Accession No. YP_355335) Core (HBcAg)*Positions 30/31-212 of SEQ ID NO: 9 e-antigen (HBeAg) *Positions 20-178of SEQ ID NO:9 HBV, Polymerase SEQ ID NO: 10 Genotype C, P (AccessionNo. ACH57822) reverse transcriptase *Positions 381-600 of SEQ ID NO: 10HBV, Surface HBsAg (L) SEQ ID NO: 11 Genotype C, S (Accession No.BAJ06646.1) Surface HBsAg (M) *Positions 120-400 of SEQ ID NO: 11Surface HBsAg (S) *Positions 175-400 of SEQ ID NO: 11 HBV, X (HBx) SEQID NO: 12 Genotype C, X (Accession No. BAJ06639.1) HBV, Precore SEQ IDNO: 13 Genotype D, C (Accession No. ADF29260.1) Core (HBcAg) *Positions30/31-212 of SEQ ID NO: 13 e-antigen (HBeAg) *Positions 20-178 of SEQ IDNO: 13 HBV, Polymerase SEQ ID NO:14 Genotype D, P (Accession No.ADD12642.1) reverse transcriptase *Positions 370-589 of SEQ ID NO: 14HBV, Surface HBsAg (L) SEQ ID NO: 15 Genotype D, S (Accession No.ACP20363.1) Surface HBsAg (M) *Positions 109-389 of SEQ ID NO: 15Surface HBsAg (S) *Positions 164-389 of SEQ ID NO: 15 HBV, X (HBx) SEQID NO: 16 Genotype D, X (Accession No. BAF47226.1) HBV, Precore SEQ IDNO: 17 Genotype E, C (Accession No. ACU25047.1) Core (HBcAg) *Positions30/31-212 of SEQ ID NO: 17 e-antigen (HBeAg) *Positions 20-178 of SEQ IDNO: 17 HBV, Polymerase SEQ ID NO: 18 Genotype E, P (Accession No.AC089764.1) reverse transcriptase *Positions 380-599 of SEQ ID NO: 18HBV, Surface HBsAg (L) SEQ ID NO: 19 Genotype E, S (Accession No.BAD91274.1) Surface HBsAg (M) *Positions 119-399 of SEQ ID NO: 19Surface HBsAg (S) *Positions 174-399 of SEQ ID NO: 19 HBV, X (HBx) SEQID NO:20 Genotype E, X (Accession No. ACU24870.1) HBV, Precore SEQ IDNO: 21 Genotype F, C (Accession No. BAB17946.1) Core (HBcAg) *Positions30/31-212 of SEQ ID NO: 21 e-antigen (HBeAg) *Positions 20-178 of SEQ IDNO: 21 HBV, Polymerase SEQ ID NO: 22 Genotype F, P (Accession No.ACD03788.2) reverse transcriptase *Positions 381-600 of SEQ ID NO: 22HBV, Surface HBsAg (L) SEQ ID NO: 23 Genotype F, S (Accession No.BAD98933.1) Surface HBsAg (M) *Positions 120-400 of SEQ ID NO: 23Surface HBsAg (S) *Positions 175-400 of SEQ ID NO: 23 HBV, SEQ ID NO: 24Genotype F, X X (HBx) (Accession No. AAM09054.1) HBV, Precore SEQ ID NO:25 Genotype G, C (Accession No. ADD62622.1) Core (HBcAg) *Positions14-194 of SEQ ID NO: 25 e-antigen (HBeAg) *Positions 4-161 of SEQ ID NO:25 HBV, Polymerase SEQ ID NO: 26 Genotype G, P (Accession No.ADD62619.1) reverse transcriptase *Positions 380-599 of SEQ ID NO: 26HBV, Surface SEQ ID NO: 27 Genotype G, S (HBsAg) (L) (Accession No.ADD62620.1) Surface HBsAg (M) *Positions 119-399 of SEQ ID NO: 27Surface HBsAg (S) *Positions 174-399 of SEQ ID NO: 27 HBV, X (HBx) SEQID NO: 28 Genotype G, X (Accession No. BAB82400.1) HBV, Precore SEQ IDNO: 29 Genotype H, C (Accession No. BAD91265.1) Core (HBcAg) *Positions30/31-212 of SEQ ID NO: 29 e-antigen (HBeAg) *Positions 20-178 of SEQ IDNO: 29 HBV, Polymerase SEQ ID NO: 30 Genotype H, P (Accession No.BAF49208.1) reverse transcriptase *Positions 381-600 of SEQ ID NO: 30HBV, Surface HBsAg (L) SEQ ID NO: 31 Genotype H, S (Accession No.BAE20065.1) Surface HBsAg (M) *Positions 120-400 of SEQ ID NO: 31Surface HBsAg (S) *Positions 175-400 of SEQ ID NO: 31 HBV, X (HBx) SEQID NO: 32 Genotype H, X (Accession No. BAF49206.1) *Position numberingis approximate and may include additional amino acids flanking eitherside of the indicated position

Hepatitis B Virus Antigens and Constructs.

One embodiment of the invention relates to novel HBV antigens and fusionproteins and recombinant nucleic acid molecules encoding these antigensand proteins. Described herein are several different novel HBV antigensfor use in a yeast-based immunotherapeutic composition or othercomposition (e.g., other immunotherapeutic or diagnostic composition)that provide one or multiple (two, three, four, five, six, seven, eight,nine or more) antigens and/or immunogenic domains from one or moreproteins, all contained within the same fusion protein and encoded bythe same recombinant nucleic acid construct (recombinant nucleic acidmolecule). The antigens used in the compositions of the inventioninclude at least one HBV protein or immunogenic domain thereof forimmunizing an animal (prophylactically or therapeutically). Thecomposition can include one, two, three, four, a few, several or aplurality of HBV antigens, including one, two, three, four, five, six,seven, eight, nine, ten, or more immunogenic domains of one, two, three,four or more HBV proteins. In some embodiments, the antigen is a fusionprotein. In one aspect of the invention, fusion protein can include twoor more proteins. In one aspect, the fusion protein can include two ormore immunogenic domains and/or two or more epitopes of one or moreproteins. An immunotherapeutic composition containing such antigens mayprovide antigen-specific immunization in a broad range of patients. Forexample, an antigen or fusion protein encompassed by the invention caninclude at least a portion of, or the full-length of, any one or moreHBV proteins selected from: HBV surface protein (also called surfaceantigen or envelope protein or HBsAg), including the large (L), middle(M) and/or small (S) forms of surface protein and/or the pre-S1 and/orpre-S2 domains thereof; HBV precore protein; HBV core protein (alsocalled core antigen or HBcAg); HBV e-antigen (also called HBeAg); HBVpolymerase (including one or both domains of the polymerase, called theRT domain and the TP domain); HBV X antigen (also called X, X antigen,or HBx); and/or any one or more immunogenic domains of any one or moreof these HBV proteins. In one embodiment, an antigen useful in animmunotherapeutic composition of the invention is from a single HBVprotein (full-length, near full-length, or portion thereof comprising atleast, one, two, three, four or more immunogenic domains of afull-length protein). In one embodiment of the invention, animmunotherapeutic composition includes one, two, three, four, five ormore individual yeast vehicles, each expressing or containing adifferent HBV antigen(s).

Combinations of HBV antigens useful in the present invention include,but are not limited to (in any order within the fusion protein):

-   -   (1) surface protein (L, M and/or S and/or any one or combination        of functional and/or immunological domains thereof, including,        but not limited to pre-S1 and/or pre-S2 and/or the hepatocyte        receptor domain of pre-S1) in combination with any one or more        of: (a) precore/core/e (precore, core, e-antigen, and/or any one        or combination of functional and/or immunological domains        thereof); (b) polymerase (full-length, RT domain, TP domain        and/or any one or combination of functional and/or immunological        domains thereof); and/or (c) X antigen (or any one or        combination of functional and/or immunological domains thereof);    -   (2) precore/core/e (precore, core, e-antigen, and/or any one or        combination of functional and/or immunological domains thereof)        in combination with any one or more of: (a) surface protein (L,        M and/or S and/or any one or combination of functional and/or        immunological domains thereof, including, but not limited to        pre-S1 and/or pre-S2 and/or the hepatocyte receptor domain of        pre-S1); (b) polymerase (full-length, RT domain, TP domain        and/or any one or combination of functional and/or immunological        domains thereof); and/or (c) X antigen (or any one or        combination of functional and/or immunological domains thereof);    -   (3) polymerase (full-length, RT domain, TP domain and/or any one        or combination of functional and/or immunological domains        thereof) in combination with any one or more of: (a) surface        protein (L, M and/or S and/or any one or combination of        functional and/or immunological domains thereof, including, but        not limited to pre-S1 and/or pre-S2 and/or the hepatocyte        receptor domain of pre-S1); (b) precore/core/e (precore, core,        e-antigen, and/or any one or combination of functional and/or        immunological domains thereof); and/or (c) X antigen (or any one        or combination of functional and/or immunological domains        thereof); or    -   (4) X antigen (or any one or combination of functional and/or        immunological domains thereof) in combination with any one or        more of: (a) surface protein (L, M and/or S and/or any one or        combination of functional and/or immunological domains thereof,        including, but not limited to pre-S1 and/or pre-S2 and/or the        hepatocyte receptor domain of pre-S1); (b) polymerase        (full-length, RT domain, TP domain and/or any one or combination        of functional and/or immunological domains thereof); and/or (c)        precore/core/e (precore, core, e-antigen, and/or any one or        combination of functional and/or immunological domains thereof).

Recombinant nucleic acid molecules and the proteins encoded thereby,including fusion proteins, as one embodiment of the invention, may beused in yeast-based immunotherapy compositions, or for any othersuitable purpose for HBV antigen(s), including in an in vitro assay, forthe production of antibodies, or in another immunotherapy composition,including another vaccine, that is not based on the yeast-basedimmunotherapy described herein. Expression of the proteins by yeast isone preferred embodiment, although other expression systems may be usedto produce the proteins for applications other than a yeast-basedimmunotherapy composition.

According to the present invention, the general use herein of the term“antigen” refers: to any portion of a protein (peptide, partial protein,full-length protein), wherein the protein is naturally occurring orsynthetically derived, to a cellular composition (whole cell, celllysate or disrupted cells), to an organism (whole organism, lysate ordisrupted cells) or to a carbohydrate, or other molecule, or a portionthereof. An antigen may elicit an antigen-specific immune response(e.g., a humoral and/or a cell-mediated immune response) against thesame or similar antigens that are encountered by an element of theimmune system (e.g., T cells, antibodies).

An antigen can be as small as a single epitope, a single immunogenicdomain or larger, and can include multiple epitopes or immunogenicdomains. As such, the size of an antigen can be as small as about 8-12amino acids (i.e., a peptide) and as large as: a full length protein, amultimer, a fusion protein, a chimeric protein, a whole cell, a wholemicroorganism, or any portions thereof (e.g., lysates of whole cells orextracts of microorganisms). In addition, antigens can includecarbohydrates, which can be loaded into a yeast vehicle or into acomposition of the invention. It will be appreciated that in someembodiments (e.g., when the antigen is expressed by the yeast vehiclefrom a recombinant nucleic acid molecule), the antigen is a protein,fusion protein, chimeric protein, or fragment thereof, rather than anentire cell or microorganism.

When the antigen is to be expressed in yeast, an antigen is of a minimumsize capable of being expressed recombinantly in yeast, and is typicallyat least or greater than 25 amino acids in length, or at least orgreater than 26, at least or greater than 27, at least or greater than28, at least or greater than 29, at least or greater than 30, at leastor greater than 31, at least or greater than 32, at least or greaterthan 33, at least or greater than 34, at least or greater than 35, atleast or greater than 36, at least or greater than 37, at least orgreater than 38, at least or greater than 39, at least or greater than40, at least or greater than 41, at least or greater than 42, at leastor greater than 43, at least or greater than 44, at least or greaterthan 45, at least or greater than 46, at least or greater than 47, atleast or greater than 48, at least or greater than 49, or at least orgreater than 50 amino acids in length, or is at least 25-50 amino acidsin length, at least 30-50 amino acids in length, or at least 35-50 aminoacids in length, or at least 40-50 amino acids in length, or at least45-50 amino acids in length. Smaller proteins may be expressed, andconsiderably larger proteins (e.g., hundreds of amino acids in length oreven a few thousand amino acids in length) may be expressed. In oneaspect, a full-length protein, or a structural or functional domainthereof, or an immunogenic domain thereof, that is lacking one or moreamino acids from the N- and/or the C-terminus may be expressed (e.g.,lacking between about 1 and about 20 amino acids from the N- and/or theC-terminus). Fusion proteins and chimeric proteins are also antigensthat may be expressed in the invention. A “target antigen” is an antigenthat is specifically targeted by an immunotherapeutic composition of theinvention (i.e., an antigen against which elicitation of an immuneresponse is desired). An “HBV antigen” is an antigen derived, designed,or produced from one or more HBV proteins such that targeting theantigen also targets the hepatitis B virus.

When referring to stimulation of an immune response, the term“immunogen” is a subset of the term “antigen”, and therefore, in someinstances, can be used interchangeably with the term “antigen”. Animmunogen, as used herein, describes an antigen which elicits a humoraland/or cell-mediated immune response (i.e., is immunogenic), such thatadministration of the immunogen to an individual mounts anantigen-specific immune response against the same or similar antigensthat are encountered by the immune system of the individual. In oneembodiment, an immunogen elicits a cell-mediated immune response,including a CD4⁺ T cell response (e.g., TH1, TH2 and/or TH17) and/or aCD8⁺ T cell response (e.g., a CTL response).

An “immunogenic domain” of a given antigen can be any portion, fragmentor epitope of an antigen (e.g., a peptide fragment or subunit or anantibody epitope or other conformational epitope) that contains at leastone epitope that acts as an immunogen when administered to an animal.Therefore, an immunogenic domain is larger than a single amino acid andis at least of a size sufficient to contain at least one epitope thatcan act as an immunogen. For example, a single protein can containmultiple different immunogenic domains Immunogenic domains need not belinear sequences within a protein, such as in the case of a humoralimmune response, where conformational domains are contemplated.

A “functional domain” of a given protein is a portion or functional unitof the protein that includes sequence or structure that is directly orindirectly responsible for at least one biological or chemical functionassociated with, ascribed to, or performed by the protein. For example,a functional domain can include an active site for enzymatic activity, aligand binding site, a receptor binding site, a binding site for amolecule or moiety such as calcium, a phosphorylation site, or atransactivation domain. Examples of HBV functional domains include, butare not limited to, the viral hepatocyte receptor domain in pre-S1, orthe reverse transcriptase domain or RNase H domain of polymerase.

A “structural domain” of a given protein is a portion of the protein oran element in the protein's overall structure that has an identifiablestructure (e.g., it may be a primary or tertiary structure belonging toand indicative of several proteins within a class or family ofproteins), is self-stabilizing and/or may fold independently of the restof the protein. A structural domain is frequently associated with orfeatures prominently in the biological function of the protein to whichit belongs.

An epitope is defined herein as a single immunogenic site within a givenantigen that is sufficient to elicit an immune response when provided tothe immune system in the context of appropriate costimulatory signalsand/or activated cells of the immune system. In other words, an epitopeis the part of an antigen that is actually recognized by components ofthe immune system, and may also be referred to as an antigenicdeterminant. Those of skill in the art will recognize that T cellepitopes are different in size and composition from B cell or antibodyepitopes, and that epitopes presented through the Class I MHC pathwaydiffer in size and structural attributes from epitopes presented throughthe Class II MHC pathway. For example, T cell epitopes presented byClass I MHC molecules are typically between 8 and 11 amino acids inlength, whereas epitopes presented by Class II MHC molecules are lessrestricted in length and may be from 8 amino acids up to 25 amino acidsor longer. In addition, T cell epitopes have predicted structuralcharacteristics depending on the specific MHC molecules bound by theepitope. Multiple different T cell epitopes have been identified invarious HBV strains and for many human HLA types, several of which areidentified in Table 5. In addition, epitopes for certain murine MHChaplotypes have been newly discovered herein and are also presented inTable 5 or in the Examples. Epitopes can be linear sequence epitopes orconformational epitopes (conserved binding regions). Most antibodiesrecognize conformational epitopes.

One exemplary embodiment of the invention relates to a fusion proteincomprising an HBV antigen that is a multi-protein HBV antigen, and inthis example, a fusion comprised of HBV large (L) surface antigen,including all of the hydrophobic transmembrane domains, and core antigen(HBcAg), described in detail below. Surface antigen and core areabundantly expressed in infected cells, are required for viralreplication, and contain multiple CD4⁺ and CD8⁺ T cell epitopes. Inaddition, these antigens, particularly surface antigen, contain knownmutation sites that can be induced by anti-viral therapy; these regionscan therefore be modified, as needed, to provide additionalimmunotherapy compositions to target the “escape” mutations. Anadditional advantage of targeting these proteins, and particularly bothproteins in a single immunotherapeutic composition, is the high degreeof conservation at the amino acid level among different HBV genotypes.Both the core and surface (L) proteins are highly conserved between HBVgenotypes A and C or between A and H, for example (see Table 4), whichare genotypes prevalent in the Americas and Asia (Table 2). The coreprotein displays a 95% amino acid identity between genotypes A and C andbetween genotypes A and H. The large (L) surface protein is also highlyconserved among the different HBV genotypes; a 90% amino acid identityexists between genotypes A and C, and 82% amino acid identity existsbetween genotypes A and H.

TABLE 4 Comparison Core Surface (L) X Polymerase HBV Genotype A 95 90 8990 vs. HBV Genotype C HBV Genotype A 95 82 79 82 vs. HBV Genotype H

Therefore, one immunotherapeutic composition designed using one HBVgenotype can be expected to induce an effective immune response againsta highly similar HBV genotype, either through direct targeting ofconserved epitopes or through epitope spreading as a result of initiallytargeting epitopes that are conserved between genotypes. Alternatively,because of the ease of producing the yeast-based immunotherapycompositions of the invention, it is straightforward to modify asequence to encode a protein, domain, or epitope from a differentgenotype, or to include in the same construct different T cell epitopesor entire domains and/or proteins from two or more different HBVgenotypes, in order to increase the wide applicability of theimmunotherapy. Examples of such HBV antigens are described in detail andexemplified below. While one immunotherapeutic composition of thepresent invention was designed to target two HBV antigens, surface andcore protein, in a single product, this approach can readily be expandedto incorporate the protein sequences of other essential, conserved, andimmunogenic HBV viral proteins to result in even broader cellular immuneresponses. Such additional fusion proteins and immunotherapeuticcompositions are described and exemplified herein.

In one embodiment of the invention, the HBV antigen(s) for use in acomposition or method of the invention are selected from HBV antigensthat have been designed to optimize or enhance their usefulness asclinical products, including in the context of a yeast-basedimmunotherapeutic composition. Such HBV antigens have been designed toproduce an HBV yeast-based immunotherapeutic product that achieves oneor more of the following goals: (1) compliance with the guidelines ofthe Recombinant DNA Advisory Committee (RAC) of the National Institutesof Health (NIH), wherein no more than two thirds (⅔) of the genome of aninfectious agent may be used in a recombinant therapeutic or vaccine;(2) inclusion of a maximized number of known T cell epitopes associatedwith immune responses to acute/self-limiting HBV infections and/orchronic HBV infections (with prioritization in one aspect based on theacute/self-limiting epitope repertoire, as discussed below); (3)maximizing or prioritizing the inclusion of immunogenic domains, andmore particularly T cell epitopes (CD4⁺ and/or CD8⁺ epitopes, anddominant and/or subdominant epitopes), that are the most conserved amongHBV genotypes and/or sub-genotypes, or that can be readily modified to aconsensus sequence or included in two or more forms to cover the mostimportant sequence differences among target genotypes; and/or (4)minimizing the number of non-natural junctions within the sequence ofthe HBV antigen in the product.

Accordingly, the invention includes, in some embodiments, modificationof HBV antigens from their naturally occurring or wild-type sequences ina given strain to meet one or more of criteria described above, as wellas to include design elements and/or antigen design criteria describedelsewhere herein. Such criteria and antigen design guidance isapplicable to yeast-based immunotherapeutics comprising HBV antigensthat are individual HBV proteins or domains, as well as HBV antigensthat include combinations of HBV proteins or domains and particularly,multi-protein antigens/fusion proteins (e.g., HBV antigens from two ormore different HBV proteins and/or domains thereof, such as combinationsof antigens from HBV surface protein, polymerase, core, e-antigen,and/or X antigen). It will be appreciated that as the complexity of theHBV antigen increases, the utilization of more of these criteria areimplemented in the construction of the antigen.

Therefore, in one embodiment of the invention, an HBV antigen useful inthe present invention as a protein or fusion protein to be expressed bya yeast includes HBV sequences encoded by nucleotide sequencesrepresenting less than two thirds (⅔) of the HBV genome (i.e., theantigens are encoded by nucleic acid sequences that in total make upless than two thirds (⅔) of the HBV genome or meet the requirements ofRAC for recombinant therapeutics and prophylactics). In one aspect, thisembodiment can be achieved by selecting HBV antigens for expression in ayeast-based immunotherapeutic that meet the RAC requirements in theirfull-length or near-full-length form (e.g., X antigen is small and whenused alone would meet the RAC requirements). In another aspect, thisembodiment is achieved by modifying the structure of the protein(s)and/or domain(s) to be included in the HBV antigen, such as by deletionof sequence to truncate proteins or remove internal sequences fromproteins, by including only selected functional, structural orimmunogenic domains of a protein, or by choosing to eliminate theinclusion of a particular protein in the antigen construct altogether.In addition, HBV yeast-based immunotherapeutics may, in one embodiment,be produced as individual antigen constructs, and then used incombination in a manner that does not contravene any restrictionsrelated to the viral genome.

In another embodiment of the invention, as discussed above, theinclusion of T cell epitopes in an HBV antigen construct (protein orfusion protein) is maximized, for example, if the HBV antigen includedin the immunotherapeutic has been modified to meet another designconsideration, such as the RAC requirement discussed above. In thisembodiment, HBV antigens useful in a yeast-based immunotherapeutic aremodified with the goal of maximizing the number of immunogenic domains,and in one aspect, the number of T cell epitopes, that are retained inthe HBV antigen. In one aspect, the inclusion of T cell epitopes in anHBV antigen is prioritized as follows:

-   -   Epitopes identified in immune responses to both        acute/self-limiting HBV infections and chronic HBV        infections>Epitopes identified in immune responses to        acute/self-limiting HBV infections>Epitopes identified in immune        responses to chronic HBV infections.        In this embodiment, without being bound by theory, the inventors        believe that immune responses from individuals who had acute or        self-limiting HBV infections may be more productive in        eliminating the viral infection than the immune responses from        individuals who have chronic HBV infections. Therefore, the        inclusion of T cell epitopes that appear to be associated with        clearance of virus in these acute or self-limiting infections        (whether dominant or sub-dominant) is prioritized as being more        likely to elicit a beneficial immune response in an immunized        individual. In addition, and again without being bound by        theory, the inventors believe that the generation of an immune        response against one or more HBV target antigens using        yeast-based immunotherapy will result in an immune response in        the immunized individual against not only the epitopes included        in the yeast-based immunotherapeutic, but also against other HBV        epitopes present in the individual. This phenomenon, referred to        as “epitope spreading” allows for the design of HBV antigens        that are focused on epitopes that appear to be most relevant to        therapeutic benefit, and the mechanism of action of a        yeast-based immunotherapeutic product then allows the immune        system to expand the immune response to cover additional target        epitopes, thereby enhancing a therapeutically productive or        beneficial immune response against HBV.

Accordingly, an HBV antigen in one embodiment comprises one or more CTLepitopes (e.g., epitopes that are recognized by a T cell receptor of acytotoxic T lymphocyte (CTL), when presented in the context of anappropriate Class I MHC molecule). In one aspect, the HBV antigencomprises one or more CD4⁺ T cell epitopes (e.g., epitopes that arerecognized by a T cell receptor of a CD4⁺ T cell, in the context of anappropriate Class II MHC molecule). In one aspect, the HBV antigencomprises one or more CTL epitopes and one or more CD4⁺ T cell epitopes.In one aspect, an HBV antigen useful in an immunotherapeutic compositionof the invention comprises one or more of the exemplary HBV CTL epitopesdescribed in Table 5. One of skill in the art will readily be able toidentify the position of the corresponding sequence for each epitope inTable 5 in a given HBV sequence of any genotype, sub-genotype, orstrain/isolate, given the guidance provided below, even though someamino acids may differ from those in Table 5. Examples of suchdifferences are illustrated in Table 5. The invention is not limited toantigens comprising these epitopes as others will be known in the artand are contemplated for use in the invention. In one embodiment, theepitope can be modified to correspond to the sequence of the epitopewithin a given genotype, sub-genotype or strain/isolate of HBV, sincethere may be one or more amino acid differences at these epitopes amonggenotypes, sub-genotypes or stain/isolates.

TABLE 5 HLA Sequence HBV Prefer- Epitope Identifier Antigen enceFLLTRILTI^(1,2,3) SEQ ID Surface A*0201 NO: 42 (e.g. positions 20-28 ofS; e.g. corresponding to positions 194-202 of SEQ ID NO: 11, positions201-209 of SEQ ID NO: 34, or positions 51-59 of SEQ ID NO: 36)GLSPTVWLSV⁵ SEQ ID Surface A*0201 NO: 43 (e.g. positions 185-194 of S;e.g. corresponding to positions 359-368 of SEQ ID NO: 11, positions366-375 of SEQ ID NO: 34, or positions 216-225** of SEQ ID NO: 36)FLPSDFFPSI^(2,3,4) SEQ ID Core A*0201 NO: 44 (e.g. positions 47-56 ofPrecore; e.g. corresponding to positions 47-56 of SEQ ID NO: 9,positions 424-433 of SEQ ID NO: 34, or positions 621-630 of SEQ ID NO:36) FLLSLGIHL¹ SEQ ID Polymerase A*0201 NO: 45 (e.g. positions 575-583of Pol; e.g. corresponding to positions 573-581 of SEQ ID NO: 10, orpositions 486-494 of SEQ ID NO:36) WLSLLVPFV^(1,3,5) SEQ ID SurfaceA*0201 NO: 46 (e.g. positions 172-180 of S; e.g. corresponding topositions 346-354 of SEQ ID NO: 11, positions 353-361^(†) of SEQ IDNO:34, or positions 203-211 of SEQ ID NO:36) KYTSFPWLL SEQ ID PolymeraseA*2402 NO: 47 (e.g. positions 756-764 of Pol; e.g. corresponding topositions 756-764 of SEQ ID NO: 10) YVNVNMGLK⁴ SEQ ID Core A*1101 NO: 48(e.g. positions 117-125 of Precore; e.g. corresponding to positions117-125 of SEQ ID NO: 9, positions 494- 502 of SEQ ID NO: 34, orpositions 691-699 of SEQ ID NO: 36) EYLVSFGVW SEQ ID Core A*2402 NO: 49(e.g. positions 146-154 of Precore; e.g. corresponding to positions 146-154 of SEQ ID NO: 9, positions 523-531 of SEQ ID NO: 34, or positions720-728 of SEQ ID NO: 36) GLSRYVARL³ SEQ ID Polymerase A*0201 NO: 50(e.g. positions 455-463 of Pol; e.g. corresponding to positions 453-461‡of SEQ ID NO: 10, or positions 366- 374 of SEQ ID NO: 36) CLFKDWEEL⁵ SEQID X A*02 NO: 51 (e.g. positions 115-123 of X; e.g. corresponding topositions 115-123^(§) of SEQ ID NO:12, or positions 900-908^(§) of SEQID NO: 36) PLGFFPDH⁵ SEQ ID Surface A*11 NO: 52 (e.g. positions 21-28 ofPre-S1; e.g. corresponding to positions 21-28 of SEQ ID NO:11, positions28-35 of SEQ ID NO: 34;, or positions 6-13 of SEQ ID NO: 36) IPIPSSWAF⁵SEQ ID Surface B*07 NO: 53 (e.g. positions 150-158 of S; e.g.corresponding to positions 324-332 of SEQ ID NO:11, positions 331-339 ofSEQ ID NO:34, or positions 181-189 of SEQ ID NO: 36) LPSDFFPSV⁵ SEQ IDCore B*51 NO: 54 (e.g. positions 48-56 of Precore; e.g. corresponding topositions 48-56^(∥) of SEQ ID NO:9, positions 425-433^(∥) of SEQ IDNO:34, or positions 619-630^(∥) of SEQ ID NO: 36) MQWNSTALH- SEQ IDSurface A*3 QALQDP⁵ NO: 55 (e.g. positions 1-15 of pre-S2; e.g.***corresponding to positions 120-134 of SEQ ID NO: 3) LLDPRVRGL⁵ SEQ IDSurface A*2 NO: 56 (e.g. positions 12-20 of pre-S2; e.g.***corresponding to positions 131-139 of SEQ ID NO: 3) SILSKTGDPV⁵ SEQID Surface A*2 NO: 57 (e.g. positions 44-53 of a pre-S2; e.g.***corresponding to positions 163-172 of SEQ ID NO: 3) VLQAGFFLL⁵ SEQ IDSurface A*2 NO: 58 (e.g. positions 14-22 of S; e.g. ***corresponding topositions 188-196 of SEQ ID NO: 3) FLLTRILTI⁵ SEQ ID Surface A*2 NO: 59(e.g. positions 20-28 of S; e.g. ***corresponding to positions 194-202of SEQ ID NO: 3) FLGGTPVCL⁵ SEQ ID Surface A*2 NO: 60 (e.g. positions41-49 of S; e.g. ***corresponding to positions 215-223 of SEQ ID NO: 3)LLCLIFLLV⁵ SEQ ID Surface A*2 NO: 61 (e.g. positions 88-96. of S; e.g***corresponding to positions 262-270 of SEQ ID NO: 3) LVLLDYQGML⁵ SEQID Surface A*2 NO: 62 (e.g. positions 95-104 of S; e.g. ***correspondingto positions 269-278 of SEQ ID NO: 3) LLDYQGMLPV⁵ SEQ ID Surface A*2 NO:63 (e.g. positions 97-106 of S; e.g. ***corresponding to positions271-280 of SEQ ID NO:3) SIVSPFIPLL⁵ SEQ ID Surface A*2 NO: 64 (e.g.positions 207-216 of S; e.g. ***corresponding to positions 381-390 ofSEQ ID NO:3) ILSPFLPLL⁵ SEQ ID Surface A*2 NO: 65 (e.g. positions208-216 of S; e.g. ***corresponding to positions 382-390 of SEQ ID NO:3) TPARVTGGVF⁵ SEQ ID Polymerase B*7 NO: 66 (e.g. positions 367-376 ofPol; e.g. ***corresponding to positions 367-376 of SEQ ID NO: 2)LWDFSQFSR⁵ SEQ ID Polymerase A*3 NO: 67 (e.g. positions 390-399 of Pol;e.g. ***corresponding to positions 390-399 of SEQ ID NO: 2) SAICSWRR⁵SEQ ID Polymerase A*3 NO: 68 (e.g. positions 533-541 of Pol; e.g.***corresponding to positions 533-541 of SEQ ID NO: 2) YMDDWLGA⁵ SEQ IDPolymerase A*2 NO: 69 (e.g. positions 551-559 . of Pol; e.g***corresponding to positions 551-559 of SEQ ID NO: 2) ALMPLYACI⁵ SEQ IDPolymerase A*2 NO: 70 (e.g. positions 655-663 . of Pol; e.g***corresponding to positions 655-663 of SEQ ID NO: 2) QAFTFSPTYK⁵ SEQID Polymerase A*3 NO: 71 (e.g. positions 667-676 of Pol; e.g.***corresponding to positions 667-676 of SEQ ID NO: 2)ATVELLSFLPSDFFPSV⁵ SEQ ID Core A*2 NO: 72 (e.g. positions 40-56 ofPrecore; e.g. ***corresponding to positions 40-56 of SEQ ID NO: 1)LPSDFFPSV⁵ SEQ ID Core B*51 NO: 73 (e.g. positions 48-56 of Precore;e.g. ***corresponding to positions 48-56 of SEQ ID NO: 1) CLTFGRETV⁵ SEQID Core A*2 NO: 74 (e.g. positions 136-144 of Precore; e.g.***corresponding to positions 136-144 of SEQ ID NO: 1) VLEYLVSFGV⁵ SEQID Core A*2 NO: 75 (e.g. positions 144-153 of Precore; e.g.***corresponding to positions 144-153 of SEQ ID NO: 1) ILSTLPETTV⁵ SEQID Core A*2 NO: 76 (e.g. positions 168-177 of Precore; e.g.***corresponding to positions 168-177 of SEQ ID NO: 1) STLPETTVVRR⁵ SEQID Core A*3 NO: 77 (e.g. positions 170-180 of Precore; e.g.***corresponding to positions 170-180 of SEQ ID NO: 1) HLSLRGLFV⁵ SEQ IDX A*2 NO: 78 (e.g. positions 52-60 of X; e.g. ***corresponding topositions 52-60 of SEQ ID NO: 4) VLHKRTLGL⁵ SEQ ID X A*2 NO: 79 (e.g.positions 92-100 of X; e.g. ***corresponding to positions 92-100 of SEQID NO: 4) GLSAMSTTDL⁵ SEQ ID X A*2 NO: 80 (e.g. positions 99-108 of X;e.g. ***corresponding to positions 99-108 of SEQ ID NO: 4) VLGGCRHKL⁵SEQ ID X A*2 NO: 81 (e.g. positions 133-141 of X; e.g. ***correspondingto positions of 133- 141 SEQ ID NO: 4) NVSIWTHK⁵ SEQ ID Polymerase A*3NO: 82 (e.g. positions 49-57 of Pol; e.g. ***corresponding to positions49-57 of SEQ ID NO: 2) KVGNFTGLY⁵ SEQ ID Polymerase A*3 NO: 83 (e.g.positions 57-65 of Pol; e.g. ***corresponding to positions 57-65 of SEQID NO: 2) GLYSSTVPV⁵ SEQ ID Polymerase A*2 NO: 84 (e.g. positions 63-71of Pol; e.g. ***corresponding to positions 63-71 of SEQ ID NO: 2)TLWKAGILYK⁵ SEQ ID Polymerase A*3 NO: 85 (e.g. positions 152-161 of Pol;e.g. ***corresponding to positions of SEQ ID NO: 2) Polymerase A*24KYTSFPWLL⁵ SEQ ID (e.g. positions 756-764 of Pol; e.g. NO: 86***corresponding to positions 758-766 of SEQ ID NO: 2) ILRGTSFVYV⁵ SEQID Polymerase A*2 NO: 87 (e.g. positions 773-782 of Pol; e.g.***corresponding to positions 773-782 of SEQ ID NO: 2) SLYADSPSV⁵ SEQ IDPolymerase A*2 NO: 88 (e.g. positions 816-824 of Pol; e.g.***corresponding to positions 816-824 of SEQ ID NO:2) KLHLYSHPI⁶ SEQ IDPolymerase A*2 NO: 135 (e.g., positions 502-510 of Pol; e.g.,***corresponding to positions 502-510 of SEQ ID NO: 2) LLVPFVQWFV^(6,7)SEQ ID Surface A*2 NO: 136 (e.g., positions 349-358 of S; e.g.,***corresponding to positions 349-358 of SEQ ID NO: 3) HLYSHPIIL⁸ SEQ IDPolymerase A*2 NO: 137 (e.g., positions 504-512 of Pol; e.g.,***corresponding to positions 504-512 of SEQ ID NO: 2) WSPQAQGIL⁹ SEQ IDSurface H-2D^(b) NO: 138 (e.g., positions 77-84 of S; e.g.,***corresponding to positions 77-84 of SEQ ID NO: 3) VLLDYQGM¹⁰ SEQ IDSurface H-2K^(b) NO: 139 (e.g., positions 270-277 of S; e.g.,***corresponding to positions 270-277 of SEQ ID NO: 3) ASVRFSWL¹⁰ SEQ IDSurface H-2K^(b) NO: 140 (e.g., positions 340-347 of S; e.g.,***corresponding to positions 340-347 of SEQ ID NO: 3) **Substitution ofan Ala for Val at position 9 of SEQ ID NO: 43; at position 225 in SEQ IDNO:36. †Substitution of Gln-Ala for Leu-Val at positions 5 and 6 of SEQID NO:46; at positions 357 and 358 in SEQ ID NO: 34. ‡Substitution ofPro for Ser at position 3 of SEQ ID NO: 50; at position 455 in SEQ IDNO: 10. §Substitution of Val for Leu at position 2 of SEQ ID NO: 51; atposition 116 in SEQ ID NO: 12 and position 901 in SEQ ID NO:36.∥Substitution of IIe for Val at position 9 of SEQ ID NO: 54; at position56 in SEQ ID NO: 9, position 433 of SEQ ID NO:34, and position 630 ofSEQ ID NO:36. ***One or more amino acid differences between the epitopesequence and the actual sequence of the corresponding larger protein ordomain may exist due to genotype, sub-genotype or strain differences,although position of the epitope within the larger protein or domain canreadily be determined. ¹Zhang et al., Journal of Hepatology 50:1163-1173 (2009) ²Lopes et al., J. Clin. Invest. 118: 1835-1845 (2008)³Boettler et al., J Virol 80(7): 3532-3540 (2006) ⁴Peng et al., Mol.Immunol. 45: 963-970 (2008) ⁵Desmond 2008; www.allelefrequencies.net orDesmond et al., Antiviral Ther. 13: 16-175 (2008) ⁶Webster et al., 2004,J. Virol. 78(11)5707-5719 ⁷Vitiello, 1997, Eur. J. Immunol. 27(3):671-678 ⁸Sette et al., 1994, J. Immunol. 153(12): 5586-5592 ⁹MurineH-2D^(b) epitope, not previously reported ¹⁰Murine H-2K^(b) epitope, notpreviously reported

In one embodiment of the invention, useful HBV antigens can include inone or more yeast-based immunotherapeutic compositions an antigencomprising one or more T cell epitopes that has been described as ordetermined to be a “dominant” epitope (i.e., a T cell epitope thatcontributes to the development of a T cell response against the wholeprotein, and/or that is among the relatively small number of T cellepitopes within the large group of possible epitopes that most likely ormost readily elicit CD4⁺ and CD8⁺ T cell responses, also referred to asan “immunodominant epitope”). In another embodiment, HBV antigens usefulin the invention can include in the same or a different or additionalyeast-based compositions, an HBV antigen comprising one or more T cellepitopes that has been described as or determined to be a “subdominant”epitope (i.e., a T cell epitope that is immunogenic, but to a lesserextent than an immunodominant epitope; the immune response generated bya sub-dominant epitope may be suppressed or outcompeted by the immuneresponse to an immunodominant epitope). For an example of this effectwith CTL responses to HBV T cell epitopes in mice, see Schirmbeck R., etal. J. immunology 168: 6253-6262, 2010; or Sette et al. J Immunology166:1389-1397, 2001. In one aspect of the invention, differentcompositions comprising immunodominant or sub-dominant epitopes could beadministered at the same site in an individual, or in one embodiment, atdifferent sites in an individual (i.e., the composition comprisingdominant epitopes being administered to one site and the compositioncomprising sub-dominant epitopes being administered to a differentsite). In some cases, a sub-dominant epitope may elicit a moretherapeutically beneficial immune response than a dominant epitope.Therefore, if administered to separate sites, it may decrease the chancethat an immune response to a dominant epitope would suppress oroutcompete an immune response to a sub-dominant epitope, therebymaximizing the immune response as a whole and maximizing the protectiveor therapeutic benefit in an individual. This approach of providingdifferent antigens in different compositions administered to differentsites in the individual can also be utilized even if all epitopes aredominant or sub-dominant Immunodominant epitopes and sub-dominantepitopes have been recognized to play a role in HBV infection and immuneresponses (see, e.g., Sette et al., 2001, supra. and Schirmbeck et al.,2002, supra).

In one embodiment of the invention, an HBV antigen useful in ayeast-based immunotherapeutic maximizes the inclusion of immunogenicdomains, and particularly, T cell epitopes, that are conserved amonggenotypes and/or sub-genotypes, and/or includes immunogenic domains fromseveral different genotypes and/or sub-genotypes and/or includesimmunogenic domains that can readily be modified to produce multipleyeast-based immunotherapeutic products that differ in some minorrespects, but are tailored to treat different individuals or populationsof individuals based on the HBV genotype(s) or sub-genotype(s) thatinfect such individuals or populations of individuals. For example, theHBV antigen can be produced based on a genotype or sub-genotype that ismost prevalent among individuals or populations of individuals to beprotected or treated, and the HBV antigen includes the most conservedimmunogenic domains from those genotypes. Alternatively or in addition,immunogenic domains can be modified to correspond to a consensussequence for that domain or epitope, or more than one version of theepitope can be included in the construct.

In any embodiment of the invention related to the design of an HBVantigen for a yeast-based immunotherapeutic composition, in one aspect,artificial junctions between segments of a fusion protein comprising HBVantigens is minimized (i.e., the inclusion of non-natural sequences islimited or minimized to the extent possible). Without being bound bytheory, it is believed that natural evolution has resulted in: i)contiguous sequences in the virus that most likely to be expressed wellin another cell, such as a yeast; and ii) an immunoproteasome in antigenpresenting cells that can properly digest and present those sequences tothe immune system. The yeast-based immunotherapeutic product of theinvention allows the host immune system to process and present targetantigens; accordingly, a fusion protein with many unnatural junctionsmay be less useful in a yeast-based immunotherapeutic as compared to onethat retains more of the natural HBV protein sequences.

In any of the HBV antigens described herein, including any of the fusionproteins, the following additional embodiments can apply. First, theN-terminal expression sequence and the C-terminal tag included in someof the fusion proteins are optional, and if used, may be selected fromseveral different sequences described elsewhere herein to improveexpression, stability, and/or allow for identification and/orpurification of the protein. Alternatively, one or both of the N- orC-terminal sequences are omitted altogether. In addition, many differentpromoters suitable for use in yeast are known in the art and areencompassed for use to express HBV antigens according to the presentinvention. Furthermore, short intervening linker sequences (e.g., 1, 2,3, 4, or 5, or larger, amino acid peptides) may be introduced betweenportions of the fusion protein for a variety of reasons, including theintroduction of restriction enzyme sites to facilitate cloning andfuture manipulation of the constructs. Finally, as discussed in detailelsewhere herein, the sequences described herein are exemplary, and maybe modified as described in detail elsewhere herein to substitute, add,or delete sequences in order to accommodate preferences for HBVgenotype, HBV subgenotype, HBV strain or isolate, or consensus sequencesand inclusion of preferred T cell epitopes, including dominant and/orsubdominant T cell epitopes. A description of several differentexemplary HBV antigens useful in the invention is provided below.

In any of the embodiments of the invention described herein, includingany embodiment related to an immunotherapeutic composition, HBV antigen,fusion protein or use of such composition, HBV antigen or fusionprotein, in one aspect, an amino acid of an HBV surface antigen usefulas an HBV antigen or in a fusion protein or an immunotherapeuticcomposition of the invention can include, but is not limited to, SEQ IDNO:3, SEQ ID NO:7, SEQ ID NO:11, positions 21-47 of SEQ ID NO:11,positions 176-400 of SEQ ID NO:11, SEQ ID NO:15, SEQ ID NO:19, SEQ IDNO:23, SEQ ID NO:27, SEQ ID NO:31, positions 9-407 of SEQ ID NO:34,positions 6-257 of SEQ ID NO:36, positions 6-257 of SEQ ID NO:41,positions 92-343 of SEQ ID NO:92, positions 90-488 of SEQ ID NO:93, SEQID NO:97, positions 90-338 of SEQ ID NO:101, positions 7-254 of SEQ IDNO:102, positions 1-249 of SEQ ID NO:107, positions 1-249 of SEQ IDNO:108, positions 1-249 of SEQ ID NO:109, positions 1-249 of SEQ IDNO:110, positions 1-399 of SEQ ID NO:112, positions 1-399 of SEQ IDNO:114, or positions 1-399 of SEQ ID NO:116, positions 1-399 of SEQ IDNO:118, positions 1-399 of SEQ ID NO:120, positions 1-399 of SEQ IDNO:122, positions 1-399 of SEQ ID NO:124, positions 1-399 of SEQ IDNO:126, positions 231-629 of SEQ ID NO:128, positions 63-461 of SEQ IDNO:130, positions 289-687 of SEQ ID NO:132, positions 289-687 of SEQ IDNO:134, or a corresponding sequence from a different HBV strain.

In any of the embodiments of the invention described herein, includingany embodiment related to an immunotherapeutic composition, HBV antigen,fusion protein or use of such composition, HBV antigen or fusionprotein, in one aspect, an amino acid of an HBV polymerase antigenuseful as an HBV antigen or in a fusion protein or an immunotherapeuticcomposition of the invention can include, but is not limited to,positions 383-602 of SEQ ID NO:2, positions 381-600 of SEQ ID NO:6,positions 381-600 of SEQ ID NO:10, positions 453 to 680 of SEQ ID NO:10,positions 370-589 of SEQ ID NO:14, positions 380-599 of SEQ ID NO:18,positions 381-600 of SEQ ID NO:22, positions 380-599 of SEQ ID NO:26,positions 381-600 of SEQ ID NO:30, positions 260 to 604 of SEQ ID NO:36,positions 7-351 of SEQ ID NO:38, positions 7-351 of SEQ ID NO:40, 260 to604 of SEQ ID NO:41, positions 346 to 690 of SEQ ID NO:92, positions90-434 of SEQ ID NO:94, SEQ ID NO:98, positions 339 to 566 of SEQ IDNO:101, positions 255 to 482 of SEQ ID NO:102, positions 250-477 of SEQID NO:107, positions 250-477 of SEQ ID NO:108, positions 250-477 of SEQID NO:109, positions 250-477 of SEQ ID NO:110, positions 582 to 809 ofSEQ ID NO:120, positions 582 to 809 of SEQ ID NO:124, positions 642 to869 of SEQ ID NO:126, positions 1 to 228 of SEQ ID NO:128, positions 1to 228 of SEQ ID NO:132, positions 61 to 288 of SEQ ID NO:134, or acorresponding sequence from a different HBV strain.

In any of the embodiments of the invention described herein, includingany embodiment related to an immunotherapeutic composition, HBV antigen,fusion protein or use of such composition, HBV antigen or fusionprotein, in one aspect, an amino acid of an HBV core antigen useful asan HBV antigen or in a fusion protein or an immunotherapeuticcomposition of the invention can include, but is not limited to,positions 31-212 of SEQ ID NO:1, positions 31-212 of SEQ ID NO:5,positions 31-212 of SEQ ID NO:9, positions 37 to 188 of SEQ ID NO:9,positions 31-212 of SEQ ID NO:13, positions 31-212 of SEQ ID NO:17,positions 31-212 of SEQ ID NO:21, positions 14-194 of SEQ ID NO:25,positions 31-212 of SEQ ID NO:29, positions 408-589 of SEQ ID NO:34,positions 605 to 786 of SEQ ID NO:36, positions 352-533 of SEQ ID NO:38,positions 160-341 of SEQ ID NO:39, positions 605-786 of SEQ ID NO:41,positions 691-872 of SEQ ID NO:92, positions 90-271 of SEQ ID NO:95, SEQID NO:99, positions 567 to 718 of SEQ ID NO:101, positions 483 to 634 ofSEQ ID NO:102, positions 2-183 of SEQ ID NO:105, positions 184-395 ofSEQ ID NO:105, positions 396-578 of SEQ ID NO:105, positions 579-761 ofSEQ ID NO:105, positions 2-183 of SEQ ID NO:106, 338-520 of SEQ IDNO:106, positions 478-629 of SEQ ID NO:107, positions 478-629 of SEQ IDNO:108, positions 478-629 of SEQ ID NO:109, positions 478-629 of SEQ IDNO:110, positions 400-581 of SEQ ID NO:112, positions 400-581 of SEQ IDNO:114, positions 400-581 of SEQ ID NO:116, positions 400-581 of SEQ IDNO:118, positions 400 to 581 of SEQ ID NO:120, positions 400 to 581 ofSEQ ID NO:122, positions 400 to 581 of SEQ ID NO:124, positions 400 to581 of SEQ ID NO:126, positions 630 to 811 of SEQ ID NO:128, positions462 to 643 of SEQ ID NO:130, positions 688 to 869 of SEQ ID NO:132,positions 688 to 869 of SEQ ID NO:134, or a corresponding sequence froma different HBV strain.

In any of the embodiments of the invention described herein, includingany embodiment related to an immunotherapeutic composition, HBV antigen,fusion protein or use of such composition, HBV antigen or fusionprotein, in one aspect, an amino acid of an HBV X antigen useful as anHBV antigen or in a fusion protein or an immunotherapeutic compositionof the invention can include, but is not limited to, SEQ ID NO:4, SEQ IDNO:8, SEQ ID NO:12, positions 2 to 154 of SEQ ID NO:12, SEQ ID NO:16,SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:32, positions 52-68followed by positions 84-126 of SEQ ID NO:4, positions 52-68 followed bypositions 84-126 of SEQ ID NO:8, positions 52-68 followed by positions84-126 of SEQ ID NO:12, positions 52-68 followed by positions 84-126 ofSEQ ID NO:16, positions 52-68 followed by positions 84-126 of SEQ IDNO:20, positions 52-68 followed by positions 84-126 of SEQ ID NO:24,positions 52-68 followed by positions 84-126 of SEQ ID NO:28, positions52-68 followed by positions 84-126 of SEQ ID NO:32, positions 787 to 939of SEQ ID NO:36, positions 7-159 of SEQ ID NO:39, positions 873-1025 ofSEQ ID NO:92, positions 90-242 of SEQ ID NO:96, SEQ ID NO:100, positions719-778 of SEQ ID NO:101, positions 635-694 of SEQ ID NO:102, positions184-337 of SEQ ID NO:106, positions 521-674 of SEQ ID NO:106, positions630-689 of SEQ ID NO:107, positions 630-689 of SEQ ID NO:108, positions630-689 of SEQ ID NO:109, positions 630-689 of SEQ ID NO:110, positions582-641 of SEQ ID NO:122, positions 810-869 of SEQ ID NO:124, positions582-641 of SEQ ID NO:126, positions 1-60 of SEQ ID NO:130, positions 229to 288 of SEQ ID NO:132, positions 1 to 60 of SEQ ID NO:134, or acorresponding sequence from a different HBV strain.

HBV Antigens Comprising Surface Antigen and Core Protein.

In one embodiment of the invention, the HBV antigen(s) for use in acomposition or method of the invention is a fusion protein comprisingHBV antigens, wherein the HBV antigens comprise or consist of HBV large(L) surface antigen or at least one immunogenic domain thereof and HBVcore protein (HBcAg) or at least one immunogenic domain thereof. In oneaspect, the HBV large (L) surface antigen and/or the HBV core protein isfull-length or near full-length. According to any embodiment of thepresent invention, reference to a “full-length” protein (or afull-length functional domain or full-length immunological domain)includes the full-length amino acid sequence of the protein orfunctional domain or immunological domain, as described herein or asotherwise known or described in a publicly available sequence. A proteinor domain that is “near full-length”, which is also a type of homologueof a protein, differs from a full-length protein or domain, by theaddition or deletion or omission of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10amino acids from the N- and/or C-terminus of such a full-length proteinor full-length domain. General reference to a protein or domain caninclude both full-length and near full-length proteins, as well as otherhomologues thereof.

In one aspect, the HBV large (L) surface antigen or the HBV core proteincomprises at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% of the linear sequence of a full-length HBV large (L)surface antigen or HBV core protein, respectively, or of the linearsequence of a portion of HBV large surface antigen that comprises thehepatocyte receptor binding portion of pre-S1 and all or a portion ofHBV small (S) surface antigen, of the linear amino acid sequencesrepresented by SEQ ID NO:97 (optimized HBV surface antigen, describedbelow), SEQ ID NO:99 (optimized core protein, described below), or acorresponding sequence from another HBV strain, as applicable. A varietyof other sequences for suitable HBV surface antigens and HBV coreantigens useful in the invention are described herein. In one aspect,the HBV large (L) surface antigen or the HBV core protein is at least80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identicalto a full-length HBV large (L) surface antigen or HBV core protein,respectively, or to another HBV surface antigen or HBV core antigendescribed herein, including the amino acid sequence represented by SEQID NO:97 (optimized HBV surface antigen, described below), SEQ ID NO:99(optimized core protein, described below), or a corresponding sequencefrom another HBV strain, as applicable.

Such a fusion protein is schematically represented in FIG. 2. Oneexample of a composition comprising such a fusion protein is describedin Example 1. In this embodiment, yeast (e.g., Saccharomyces cerevisiae)were engineered to express various HBV surface-core fusion proteins asshown in FIG. 2 under the control of the copper-inducible promoter,CUP1, or the TEF2 promoter. In each case, the HBV fusion protein was asingle polypeptide with the following sequence elements fused in framefrom N- to C-terminus, represented by SEQ ID NO:34: (1) an N-terminalpeptide to impart resistance to proteasomal degradation and stabilizeexpression (e.g., positions 1 to 6 of SEQ ID NO:34); 2) a two amino acidspacer to introduce a SpeI restriction enzyme site; 3) the amino acidsequence of a near full-length (minus position 1) HBV genotype C large(L) surface antigen (e.g., positions 9 to 407 of SEQ ID NO:34 orpositions 2-400 of SEQ ID NO:11 (which differs from SEQ ID NO:34 atpositions 350-351 of SEQ ID NO:11, where a Leu-Val sequence in SEQ IDNO:11 is replaced with a Gln-Ala sequence at positions 357-358 of SEQ IDNO:34)); 4) the amino acid sequence of an HBV core antigen (e.g.,positions 31-212 of SEQ ID NO:9 or positions 408 to 589 of SEQ IDNO:34); and 5) a hexahistidine tag (e.g., positions 590-595 of SEQ IDNO:34). Positions 28-54 of SEQ ID NO:34 comprise the hepatocyte receptorportion of large (L) surface protein. SEQ ID NO:34 contains multipleepitopes or domains that are believed to enhance the immunogenicity ofthe fusion protein. For example, positions 209-220, positions 389-397,positions 360-367, and positions 499-506, with respect to SEQ ID NO:34,comprise known MHC Class I binding and/or CTL epitopes. Positions305-328 of SEQ ID NO:34 comprise an antibody epitope. A nucleic acidsequence encoding the fusion protein of SEQ ID NO:34 (codon optimizedfor yeast expression) is represented herein by SEQ ID NO:33. Ayeast-based immunotherapy composition expressing this fusion protein isalso referred to herein as GI-13002.

The amino acid segments used in any of the fusion proteins describedherein can be modified by the use of additional amino acids flankingeither end of any domain; the descriptions provided herein areexemplary. For example, a fusion protein according to this embodimentcan include 1) the amino acid sequence of a near full-length (minusposition 1) HBV genotype C large (L) surface antigen (e.g., positions2-400 of SEQ ID NO:11 or positions 9 to 407 of SEQ ID NO:34); and 2) theamino acid sequence of an HBV core antigen (e.g., positions 31-212 ofSEQ ID NO:9 or positions 408 to 589 of SEQ ID NO:34), and utilize no N-or C-terminal sequences, or utilize different N- or C-terminal sequencesand/or linkers or no linkers between HBV sequences. In one embodiment,instead of the N-terminal peptide represented by positions 1-6 of SEQ DINO:34, an N-terminal peptide represented by SEQ ID NO:89 or SEQ ID NO:90is utilized, followed by the remainder of the fusion protein, includingor not including the hexahistidine C-terminal tag. The fusion proteinmay also include one, two, three, four, five, six, or more linker(spacer) amino acids between HBV proteins or domains. The same alternateembodiments apply to any fusion protein or HBV antigen construct used inthe invention as described herein.

The HBV sequences used to design this fusion protein and many of theothers described and/or exemplified herein are based on isolates of aparticular HBV genotype (e.g., genotype A, B, C, or D). However, it isan embodiment of the invention to add to or substitute into any portionof an HBV antigen described herein that is based on or derived from oneparticular genotype, sub-genotype, or strain, a corresponding sequence,or even a single or small amino acid substitution, insertion or deletionthat occurs in a corresponding sequence, from any other HBV genotype(s),sub-genotype(s), or strain(s). In one embodiment, an HBV antigen can beproduced by substituting an entire sequence(s) of an HBV antigendescribed herein with the corresponding sequence(s) from one or moredifferent HBV genotypes, sub-genotypes or strain/isolates. Adding to orsubstituting a sequence from one HBV genotype or sub-genotype foranother, for example, allows for the customization of theimmunotherapeutic composition for a particular individual or populationof individuals (e.g., a population of individuals within a given countryor region of a country, in order to target the HBV genotype(s) that ismost prevalent in that country or region of the country). Similarly, itis also an embodiment of the invention to use all or a portion of aconsensus sequence derived from, determined from, or published for, agiven HBV strain, genotype or subtype to make changes in the sequence ofa given HBV antigen to more closely or exactly correspond to theconsensus sequence. According to the present invention and as generallyunderstood in the art, a “consensus sequence” is typically a sequencebased on the most common nucleotide or amino acid at a particularposition of a given sequence after multiple sequences are aligned.

As a particular example of the above-mentioned types of modifications,an HBV antigen can be modified to change a T cell epitope in a givensequence from one isolate to correspond more closely or exactly with a Tcell epitope from a different isolate, or to correspond more closely orexactly with a consensus sequence for the T cell epitope. Such T cellepitopes can include dominant epitopes and/or sub-dominant epitopes.Indeed, according to the invention, HBV antigens can be designed thatincorporate consensus sequences from a variety of HBV genotypes and/orsubtypes, or mixtures of sequences from different HBV genotypes and/orsubtypes. Alignments of major HBV proteins across exemplary sequencesfrom each of the major known genotypes can be readily generated usingpublicly available software, which will inform the generation ofconsensus sequences, for example. Furthermore, consensus sequences formany HBV proteins have been published. Since there is a high degree ofconservation at the amino acid level among different HBV genotypes,sub-genotypes and strains, it is straightforward to use thecorresponding portions of HBV proteins from genotypes, sub-genotypes orstrains other than those exemplified herein to create HBV antigenshaving a similar or the same overall structure as those describedherein. Examples of such modifications are illustrated and exemplifiedherein.

By way of example, there can be minor differences among sequences of thesame protein even within the same serotype and genotype (i.e., due tostrain or isolate variations), although such differences in sequenceidentity will typically be less than 20% across the full length of thesequences being compared (i.e., the sequences will be at least 80%identical), and more typically, the sequences will be at least 85%identical, 90% identical, 91% identical, 92% identical, 93% identical,94% identical, 95% identical, 96% identical, 97% identical, 98%identical, 99% identical, or 100% identical, over the full length of thecompared sequences. For example, in the fusion protein described above(SEQ ID NO:34), the sequence for the large (L) surface antigen used inthe fusion (positions 9-407 of SEQ ID NO:34) is from an HBV genotype Cisolate, and is about 99% identical to positions 2-400 of SEQ ID NO:11,which is also from large (L) surface antigen from an HBV genotype Cisolate (i.e., there are two different amino acids, at positions 350-351of SEQ ID NO:11 (Gln-Ala) as compared to positions 357-358 of SEQ IDNO:34 (Leu-Val). However, either sequence is suitable for use in afusion protein described herein, as are sequences from other HBVstrains. Accordingly, in one embodiment, the sequences utilized in anyof the HBV antigens described herein, including any of the fusionproteins described herein, can include the corresponding sequences fromone or more different HBV genotypes, sub-genotypes, or strains.

The above-described utilization of consensus sequences and individualHBV genotypes has been applied to various HBV antigens described herein.For example, consensus sequence design has been applied to the fusionprotein described above with reference to SEQ ID NO:34, which containsHBV surface proteins and HBV core proteins. Example 7 describesadditional fusion proteins that are similar in design to the fusionprotein represented by SEQ ID NO:34, but that are based on a consensussequence for HBV genotypes A, B, C and D, respectively. A fusion proteincomprising HBV surface and core proteins that is based on a consensussequence for HBV genotype A, which is also schematically illustrated inFIG. 2, is a single polypeptide with the following sequence elementsfused in frame from N- to C-terminus, represented by SEQ ID NO:112(optional sequences that are not HBV sequences are not included in thebase sequence of SEQ ID NO:112, but may be added to this sequence as inthe construct described in Example 7): (1) optionally, an N-terminalpeptide that is a synthetic N-terminal peptide designed to impartresistance to proteasomal degradation and stabilize expressionrepresented by SEQ ID NO:37, which may be substituted by an N-terminalpeptide represented by SEQ ID NO:89, SEQ ID NO:90, or another N-terminalpeptide suitable for use with a yeast-based immunotherapeutic asdescribed herein; (2) optionally, a linker peptide of from one to threeor more amino acids linker sequences of one, two, three or more aminoacids, such as the two amino acid linker of Thr-Ser; (3) the amino acidsequence of a near full-length (minus position 1) a consensus sequencefor HBV genotype A large (L) surface antigen represented by positions 1to 399 of SEQ ID NO:112; (4) the amino acid sequence of a consensussequence for HBV genotype A core antigen represented by positions 400 to581 of SEQ ID NO:112; and (5) optionally, a hexahistidine tag. A nucleicacid sequence encoding the fusion protein of SEQ ID NO:112 (codonoptimized for yeast expression) is represented herein by SEQ ID NO:111.A yeast-based immunotherapy composition expressing this fusion proteinis also referred to herein as GI-13006.

Example 7 also describes a fusion protein that is similar in design tothe fusion protein represented by SEQ ID NO:34, but that is based on aconsensus sequence for HBV genotype B. This fusion protein, which isalso schematically illustrated in FIG. 2, is a single polypeptide withthe following sequence elements fused in frame from N- to C-terminus,represented by SEQ ID NO:114 (optional sequences that are not HBVsequences are not included in the base sequence of SEQ ID NO:114, butmay be added to this sequence as in the construct described in Example7): (1) optionally, an N-terminal peptide that is a synthetic N-terminalpeptide designed to impart resistance to proteasomal degradation andstabilize expression represented by SEQ ID NO:37, which may besubstituted by an N-terminal peptide represented by SEQ ID NO:89, SEQ IDNO:90, or another N-terminal peptide suitable for use with a yeast-basedimmunotherapeutic as described herein; (2) optionally, a linker peptideof from one to three or more amino acids linker sequences of one, two,three or more amino acids, such as the two amino acid linker of Thr-Ser;(3) the amino acid sequence of a near full-length (minus position 1) aconsensus sequence for HBV genotype B large (L) surface antigenrepresented by positions 1 to 399 of SEQ ID NO:114; (4) the amino acidsequence of a consensus sequence for HBV genotype B core antigenrepresented by positions 400 to 581 of SEQ ID NO:114; and (5)optionally, a hexahistidine tag. A nucleic acid sequence encoding thefusion protein of SEQ ID NO:114 (codon optimized for yeast expression)is represented herein by SEQ ID NO:113. A yeast-based immunotherapycomposition expressing this fusion protein is also referred to herein asGI-13007.

Example 7 describes a fusion protein that is similar in design to thefusion protein represented by SEQ ID NO:34, but that is based on aconsensus sequence for HBV genotype C. This fusion protein, which isalso schematically illustrated in FIG. 2, is a single polypeptide withthe following sequence elements fused in frame from N- to C-terminus,represented by SEQ ID NO:116 (optional sequences that are not HBVsequences are not included in the base sequence of SEQ ID NO:116, butmay be added to this sequence as in the construct described in Example7): (1) optionally, an N-terminal peptide that is a synthetic N-terminalpeptide designed to impart resistance to proteasomal degradation andstabilize expression represented by SEQ ID NO:37, which may besubstituted by an N-terminal peptide represented by SEQ ID NO:89, SEQ IDNO:90, or another N-terminal peptide suitable for use with a yeast-basedimmunotherapeutic as described herein; (2) optionally, a linker peptideof from one to three or more amino acids linker sequences of one, two,three or more amino acids, such as the two amino acid linker of Thr-Ser;(3) the amino acid sequence of a near full-length (minus position 1) aconsensus sequence for HBV genotype C large (L) surface antigenrepresented by positions 1 to 399 of SEQ ID NO:116; (4) the amino acidsequence of a consensus sequence for HBV genotype C core antigenrepresented by positions 400 to 581 of SEQ ID NO:116; and (5)optionally, a hexahistidine tag. A nucleic acid sequence encoding thefusion protein of SEQ ID NO:116 (codon optimized for yeast expression)is represented herein by SEQ ID NO:115. A yeast-based immunotherapycomposition expressing this fusion protein is also referred to herein asGI-13008.

Example 7 describes a fusion protein that is similar in design to thefusion protein represented by SEQ ID NO:34, but that is based on aconsensus sequence for HBV genotype D. This fusion protein, which isalso schematically illustrated in FIG. 2, is a single polypeptide withthe following sequence elements fused in frame from N- to C-terminus,represented by SEQ ID NO:118 (optional sequences that are not HBVsequences are not included in the base sequence of SEQ ID NO:118, butmay be added to this sequence as in the construct described in Example7): (1) optionally, an N-terminal peptide that is a synthetic N-terminalpeptide designed to impart resistance to proteasomal degradation andstabilize expression represented by SEQ ID NO:37, which may besubstituted by an N-terminal peptide represented by SEQ ID NO:89, SEQ IDNO:90, or another N-terminal peptide suitable for use with a yeast-basedimmunotherapeutic as described herein; (2) optionally, a linker peptideof from one to three or more amino acids linker sequences of one, two,three or more amino acids, such as the two amino acid linker of Thr-Ser;(3) the amino acid sequence of a near full-length (minus position 1) aconsensus sequence for HBV genotype D large (L) surface antigenrepresented by positions 1 to 399 of SEQ ID NO:118; (4) the amino acidsequence of a consensus sequence for HBV genotype D core antigenrepresented by positions 400 to 581 of SEQ ID NO:118; and (5)optionally, a hexahistidine tag. The amino acid sequence of a completefusion protein described in Example 7 comprising SEQ ID NO:118 andincluding the N- and C-terminal peptides and linkers is representedherein by SEQ ID NO:151. A nucleic acid sequence encoding the fusionprotein of SEQ ID NO:118 or SEQ ID NO:151 (codon optimized for yeastexpression) is represented herein by SEQ ID NO:117. A yeast-basedimmunotherapy composition expressing this fusion protein is alsoreferred to herein as GI-13009.

HBV Antigens Comprising Surface Antigen, Core Protein, Polymerase and XAntigen.

In one embodiment of the invention, the HBV antigen(s) for use in acomposition or method of the invention is a fusion protein comprisingHBV antigens, wherein the HBV antigens comprise or consist of: the HBVsurface antigen (large (L), medium (M) or small (S)) or at least onestructural, functional or immunogenic domain thereof), HBV polymerase orat least one structural, functional or immunogenic domain thereof, theHBV core protein (HBcAg) or HBV e-antigen (HBeAg) or at least onestructural, functional or immunogenic domain thereof, and the HBV Xantigen (HBx) or at least one structural, functional or immunogenicdomain thereof. In one aspect, any one or more of the HBV surfaceantigen, HBV polymerase, HBV core protein, HBV e-antigen, HBV X antigen,or domain thereof, is full-length or near full-length. In one aspect,any one or more of the HBV surface antigen, HBV polymerase, HBV coreprotein, HBV e-antigen, HBV X antigen, or domain thereof comprises atleast 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% ofthe linear sequence of a full-length HBV surface antigen, HBVpolymerase, HBV core protein, HBV e-antigen, HBV X antigen, or domainthereof, respectively, or of the linear amino acid sequences representedby SEQ ID NO:97 (optimized HBV surface antigen, described below), SEQ IDNO:98 (optimized HBV polymerase, described below), SEQ ID NO:99(optimized core protein, described below), SEQ ID NO:100 (optimized Xantigen, described below), or a corresponding sequence from another HBVstrain, as applicable. In one aspect, any one or more of the HBV surfaceantigen, HBV polymerase, HBV core protein, HBV e-antigen, HBV X antigen,or domain thereof is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identical to a full-length HBV surface antigen,HBV polymerase, HBV core protein, HBV e-antigen, HBV X antigen, ordomain thereof, respectively, or to the amino acid sequences representedby SEQ ID NO:97 (optimized HBV surface antigen, described below), SEQ IDNO:98 (optimized HBV polymerase, described below), SEQ ID NO:99(optimized core protein, described below), or SEQ ID NO:100 (optimized Xantigen, described below), or a corresponding sequence from another HBVstrain, as applicable. A variety of suitable and exemplary sequences forHBV surface antigens, HBV polymerase antigens, HBV core antigens, andHBV X antigens are described herein.

In one embodiment of the invention, the HBV antigen(s) for use in acomposition or method of the invention is a fusion protein comprisingHBV antigens, wherein the HBV antigens comprise or consist of: thehepatocyte receptor portion of Pre-S1 of the HBV large (L) surfaceantigen or at least one immunogenic domain thereof, an HBV small (S)surface antigen (HBsAg) or at least one immunogenic domain thereof, thereverse transcriptase (RT) domain of HBV polymerase or at least oneimmunogenic domain thereof, the HBV core protein (HBcAg) or at least oneimmunogenic domain thereof, and the HBV X antigen (HBx) or at least oneimmunogenic domain thereof. In one aspect, any one or more of thehepatocyte receptor portion of Pre-S1 of the HBV large (L) surfaceantigen, the HBV small (S) surface antigen, the RT domain of HBVpolymerase, the HBV core protein, X antigen, or domain thereof, isfull-length or near full-length. In one aspect, any one or more of thehepatocyte receptor portion of Pre-S1 of the HBV large (L) surfaceantigen, the HBV small (S) surface antigen, the RT domain of HBVpolymerase, the HBV core protein, X antigen, or domain thereof,comprises at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% of the linear sequence of a full-length Pre-S1 of the HBVlarge (L) surface antigen, the HBV small (S) surface antigen, the RTdomain of HBV polymerase, the HBV core protein, X antigen, or domainthereof, respectively. In one aspect, any one or more of the hepatocytereceptor portion of Pre-S1 of the HBV large (L) surface antigen, the HBVsmall (S) surface antigen, the RT domain of HBV polymerase, the HBV coreprotein, X antigen, or domain thereof is at least 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a full-lengthhepatocyte receptor portion of Pre-S1 of the HBV large (L) surfaceantigen, the HBV small (S) surface antigen, the RT domain of HBVpolymerase, the HBV core protein, X antigen, or domain thereof,respectively.

Such a fusion protein is schematically represented in FIG. 3. An exampleof a composition comprising this fusion protein is described in Example2. In this embodiment, yeast (e.g., Saccharomyces cerevisiae) wereengineered to express various HBV fusion proteins as schematically shownin FIG. 3 under the control of the copper-inducible promoter, CUP1, orthe TEF2 promoter. In one case, the fusion protein is a singlepolypeptide with the following sequence elements fused in frame from N-to C-terminus, represented by SEQ ID NO:36: (1) an N-terminal peptide toimpart resistance to proteasomal degradation and stabilize expression(e.g., positions 1 to 5 of SEQ ID NO:36); 2) the amino acid sequence ofan HBV genotype C hepatocyte receptor domain of the pre-S1 portion ofHBV large (L) surface protein (unique to L) (e.g., positions 21-47 ofSEQ ID NO:11 or positions 6 to 32 of SEQ ID NO:36); 3) the amino acidsequence of a full-length HBV genotype C small (S) surface antigen(e.g., positions 176 to 400 of SEQ ID NO:11 or positions 33 to 257 ofSEQ ID NO:36); 4) a two amino acid spacer/linker to facilitate cloningand manipulation of the sequences (e.g., positions 258 and 259 of SEQ IDNO:36); 5) the amino acid sequence of a portion of the HBV genotype Cpolymerase including the reverse transcriptase domain (e.g., positions247 to 691 of SEQ ID NO:10 or positions 260 to 604 of SEQ ID NO:36); 6)an HBV genotype C core protein (e.g., positions 31-212 of SEQ ID NO:9 orpositions 605 to 786 of SEQ ID NO:36); 7) the amino acid sequence of anHBV genotype C X antigen (e.g., positions 2 to 154 of SEQ ID NO:12 orpositions 787 to 939 of SEQ ID NO:36); and 8) a hexahistidine tag (e.g.,positions 940 to 945 of SEQ ID NO:36). A nucleic acid sequence encodingthe fusion protein of SEQ ID NO:36 (codon optimized for yeastexpression) is represented herein by SEQ ID NO:35. A yeast-basedimmunotherapy composition expressing this fusion protein is referred toherein as GI-13005.

In one alternate example of this embodiment, a fusion protein accordingto the embodiment described above or that below can include 1) the aminoacid sequence of an HBV genotype C hepatocyte receptor domain of thepre-S1 portion of HBV large (L) surface protein (unique to L) (e.g.,positions 21-47 of SEQ ID NO:11 or positions 6 to 32 of SEQ ID NO:36);2) the amino acid sequence of a full-length HBV genotype C small (S)surface antigen (e.g., positions 176 to 400 of SEQ ID NO:11 or positions33 to 257 of SEQ ID NO:36); 3) the amino acid sequence of a portion ofthe HBV genotype C polymerase including the reverse transcriptase domain(e.g., positions 247 to 691 of SEQ ID NO:10 or positions 260 to 604 ofSEQ ID NO:36); 4) an HBV genotype C core protein (e.g., positions 31-212of SEQ ID NO:9 or positions 605 to 786 of SEQ ID NO:36); and 5) theamino acid sequence of an HBV genotype C X antigen (e.g., positions 2 to154 of SEQ ID NO:12 or positions 787 to 939 of SEQ ID NO:36), andutilize no N- or C-terminal sequences, or utilize different N- orC-terminal sequences, and/or use linkers or no linkers between HBVsequences.

In one embodiment, instead of the N-terminal peptide represented bypositions 1-5 of SEQ DI NO:36, an N-terminal peptide represented by SEQID NO:89 or SEQ ID NO:90 is utilized (or a homologue thereof), followedby the remainder of the fusion protein as described. Example 2 describessuch a fusion protein, which is also illustrated by the schematicdepiction of the construct in FIG. 3. In this embodiment, yeast (e.g.,Saccharomyces cerevisiae) were again engineered to express various HBVfusion proteins as schematically shown in FIG. 3 under the control ofthe copper-inducible promoter, CUP1, or the TEF2 promoter. In thissecond case, the fusion protein is a single polypeptide with thefollowing sequence elements fused in frame from N- to C-terminus,represented by SEQ ID NO:92: (1) an N-terminal peptide to impartresistance to proteasomal degradation and stabilize or enhanceexpression (SEQ ID NO:89, positions 1 to 89 of SEQ ID NO:92); 2) a twoamino acid spacer/linker (Thr-Ser) to facilitate cloning andmanipulation of the sequences (positions 90 to 91 of SEQ ID NO:92); 3)the amino acid sequence of an HBV genotype C hepatocyte receptor domainof the pre-S1 portion of HBV large (L) surface protein (unique to L)(e.g., positions 21-47 of SEQ ID NO:11 or positions 92 to 118 of SEQ IDNO:92); 4) the amino acid sequence of a full-length HBV genotype C small(S) surface antigen (e.g., positions 176 to 400 of SEQ ID NO:11 orpositions 119 to 343 of SEQ ID NO:92); 5) a two amino acid spacer/linker(Leu-Glu) to facilitate cloning and manipulation of the sequences (e.g.,positions 344 to 345 of SEQ ID NO:92); 6) the amino acid sequence of aportion of the HBV genotype C polymerase including the reversetranscriptase domain (e.g., positions 247 to 691 of SEQ ID NO:10 orpositions 346 to 690 of SEQ ID NO:92); 7) an HBV genotype C core protein(e.g., positions 31-212 of SEQ ID NO:9 or positions 691 to 872 of SEQ IDNO:92); 8) the amino acid sequence of an HBV genotype C X antigen (e.g.,positions 2 to 154 of SEQ ID NO:12 or positions 873 to 1025 of SEQ IDNO:92); and 9) a hexahistidine tag (e.g., positions 1026 to 1031 of SEQID NO:92). A nucleic acid sequence encoding the fusion protein of SEQ IDNO:92 (codon-optimized for expression in yeast) is represented herein bySEQ ID NO:91. A yeast-based immunotherapy composition expressing thisfusion protein is referred to herein as GI-13004.

SEQ ID NO:36 and SEQ ID NO:92 contain multiple epitopes or domains thatare believed to enhance the immunogenicity of the fusion protein,including several described above for SEQ ID NO:34. In addition, thereverse transcriptase domain used in this fusion protein containsseveral amino acid positions that are known to become mutated as adrug-resistance response to treatment with various anti-viral drugs, andtherefore, any one or more of these may be mutated in this fusionprotein in order to provide a therapeutic or prophylacticimmunotherapeutic that targets specific drug resistance (escape)mutations. These amino acid positions are, with respect to SEQ ID NO:36,at amino acid position: 432 (Val, known to mutate to a Leu afterlamivudine therapy); position 439 (Leu, known to mutate to a Met afterlamivudine therapy); position 453 (Ala, known to mutate to a Thr aftertenofovir therapy); position 463 (Met, known to mutate to an Ile or Valafter lamivudine therapy); and position 495 (Asn, known to mutate to Thrafter adefovir therapy). These amino acid positions are, with respect toSEQ ID NO:92, at amino acid position: 518 (Val, known to mutate to a Leuafter lamivudine therapy); position 525 (Leu, known to mutate to a Metafter lamivudine therapy); position 539 (Ala, known to mutate to a Thrafter tenofovir therapy); position 549 (Met, known to mutate to an Ileor Val after lamivudine therapy); and position 581 (Asn, known to mutateto Thr after adefovir therapy). Additional drug resistance mutationsthat are identified or that have been identified can be added, asdesired, to create additional immunotherapeutics targeting suchmutations, using the guidance provided herein.

In one embodiment of the invention, the valine at position 901 in SEQ IDNO:36 or the valine at position 987 of SEQ ID NO:92 (or the valine atposition 116 of SEQ ID NO:12 or in any X antigen or domain thereofcontaining this corresponding position) is substituted with a leucine,to create the T cell epitope identified as SEQ ID NO:51 (see Table 5).

As discussed above, the invention includes the modification of HBVantigens from their naturally occurring or wild-type sequences forinclusion in a yeast-based immunotherapeutic that improve the clinicalutility or meet required criteria for therapeutics or prophylacticsrelated to infectious agents. By way of example, the followingdiscussion and Examples 5-8 describe the design and construction ofyeast-based immunotherapeutics that takes into consideration one or morecriteria of RAC requirements, maximization of immunogenic domainsassociated with the most beneficial immune responses, maximization ofconserved T cell epitopes, utilization of consensus sequences for aparticular HBV genotype, and/or minimization of artificial junctionswithin the HBV antigen. For example, the following yeast-basedimmunotherapeutic composition exemplifies an HBV fusion protein meetingthe requirements of the goals specified above, and comprising portionsof each of the HBV major proteins: HBV surface antigen, polymerase, coreand X antigen. To design this fusion protein, individual HBV antigenswithin the fusion were optimized or modified to reduce the size of thesegments in the protein (e.g., to ensure that the protein representedless than ⅔ of the HBV genome), as well as to maximize the inclusion ofT cell epitopes that have been associated with an immune response inacute/self-limiting HBV infection and/or chronic HBV infection, tomaximize conserved epitopes, and to minimize non-natural sequences. Oneof skill in the art using this guidance can produce alternate optimizedHBV proteins for use in an HBV antigen of the invention.

As described in more detail in Example 5, to construct an HBV surfaceantigen segment, a full-length large (L) surface antigen protein fromHBV genotype C was reduced in size by truncation of the N- andC-terminal sequences, while maximizing the inclusion of known MHC T cellepitopes, using the prioritization for inclusion of T cell epitopesassociated with acute/self-limiting infections. The resulting surfaceantigen segment is represented by SEQ ID NO:97.

To construct the segment of the fusion protein comprising HBV polymerase(see Example 5), substantial portions of a full-length polymerase fromHBV genotype C were eliminated by focusing on inclusion of the activesite domain (from the RT domain), which is the most conserved region ofthe protein among HBV genotypes and isolates, and which includes severalsites where drug resistance mutations have been known to occur. The HBVpolymerase segment was designed to maximize known T cell epitopes, usingthe prioritization strategy discussed above, and to modify one of the Tcell epitopes to correspond exactly to a known T cell epitope thatdiffered by a single amino acid. The resulting HBV polymerase antigensegment is represented by SEQ ID NO:98.

To construct the segment of the fusion protein comprising HBV Coreantigen (see Example 5), a full-length Core protein from HBV genotype Cwas modified to reduce the size of the protein while maximizing thenumber of T cell epitopes by inclusion and by modification of sequenceto created perfect matches to certain known T cell epitopes. Inaddition, sequence was removed that contained exceptionally positivelycharged C-terminus which may be toxic to yeast by competitiveinterference with natural yeast RNA binding proteins which often arearginine rich (positively charged). The resulting HBV Core antigensegment is represented by SEQ ID NO:99.

To construct the segment of the fusion protein comprising HBV X antigen(see Example 5), a full-length X antigen from HBV genotype C wastruncated to reduce the size of the protein, while maximizing theretention of most of the known T cell epitopes. Single amino acidchanges were also introduced to correspond to the published T cellepitope sequences, and sequence flanking the T cell epitopes at the endsof the segment was retained to facilitate efficient processing andpresentation of the correct epitopes by an antigen presenting cell. Theresulting HBV X antigen segment is represented by SEQ ID NO:100.

Finally, as described in Example 5, a complete fusion protein wasconstructed by linking the four HBV segments described above to form asingle protein optimized for clinical use. Two different exemplaryfusion proteins were created, each with a different N-terminal peptideadded to enhance and/or stabilize expression of the fusion protein inyeast. As described previously herein with respect to all of the otherproteins used in a yeast-based immunotherapeutic compositions describedherein, the N-terminal peptide can be replaced with a differentsynthetic or natural N-terminal peptide or with a homologue thereof, orthe N-terminal peptide can be omitted altogether and a methionineincluded at position one. In addition, linker sequences of one, two,three or more amino acids may be added between segments of the fusionprotein, if desired. For example, a two amino acid linker sequence suchas Thr-Ser may be inserted between the N-terminal peptide and the firstHBV antigen in the fusion protein, and/or between two HBV antigens inthe fusion protein. Also, while these constructs were designed using HBVproteins from genotype C as the backbone, any other HBV genotype,sub-genotype, or HBV proteins from different strains or isolates can beused to design the protein segments. In one aspect, consensus sequencesfrom a given HBV genotype can be used to design or form the proteinsegments, as described in additional fusion proteins below. Finally, ifone or more segments are excluded from the fusion protein as describedherein, then the sequence from the remaining segments can be expanded inlength, if desired, to include additional T cell epitopes and/orflanking regions of the remaining proteins.

Example 5 describes an HBV fusion protein, which is also illustrated bythe schematic depiction of the construct in FIG. 3, that is a singlepolypeptide with the following sequence elements fused in frame from N-to C-terminus, represented by SEQ ID NO:101: (1) an N-terminal peptidethat is an alpha factor prepro sequence, to impart resistance toproteasomal degradation and stabilize expression represented by SEQ IDNO:89 (positions 1-89 of SEQ ID NO:101); (2) an optimized portion of anHBV large (L) surface antigen represented by SEQ ID NO:97 (positions 90to 338 of SEQ ID NO:101, e.g., corresponding to positions 120 to 368 ofSEQ ID NO:11 plus optimization of epitopes); (3) an optimized portion ofthe reverse transcriptase (RT) domain of HBV polymerase represented bySEQ ID NO:98 (positions 339 to 566 of SEQ ID NO:101, e.g., correspondingto positions 453 to 680 of SEQ ID NO:10 plus optimization of epitopes);(4) an optimized portion of HBV Core protein represented by SEQ ID NO:99(positions 567 to 718 of SEQ ID NO:101 e.g., corresponding to positions37 to 188 of SEQ ID NO:9 plus optimization of epitopes); (5) anoptimized portion of HBV X antigen represented by SEQ ID NO:100(positions 719 to 778 of SEQ ID NO:101, e.g., corresponding to positions52 to 127 of SEQ ID NO:12 plus optimization of epitopes); and (6) ahexahistidine tag (e.g., positions 779 to 784 of SEQ ID NO:101). In oneembodiment, the linker sequence of threonine (Thr or T)-serine (Ser orS) is used between the N-terminal peptide of SEQ ID NO:89 and the firstHBV protein (optimized portion of HBV large surface antigen), therebyextending the total length of SEQ ID NO:101 by two amino acids.

Example 5 also describes a fusion protein, which is also illustrated bythe schematic depiction of the construct in FIG. 3, that is a singlepolypeptide with the following sequence elements fused in frame from N-to C-terminus, represented by SEQ ID NO:102: (1) an N-terminal peptidethat is a synthetic N-terminal peptide designed to impart resistance toproteasomal degradation and stabilize expression represented by SEQ IDNO:37 (positions 1-6 of SEQ ID NO:102); (2) an optimized portion of anHBV large (L) surface antigen represented by positions 2 to 248 of SEQID NO:97 (positions 7 to 254 of SEQ ID NO:102, e.g., corresponding topositions 120 to 368 of SEQ ID NO:11 plus optimization of epitopes); (3)an optimized portion of the reverse transcriptase (RT) domain of HBVpolymerase represented by SEQ ID NO:98 (positions 255 to 482 of SEQ IDNO:102, e.g., corresponding to positions 453 to 680 of SEQ ID NO:10 plusoptimization of epitopes); (4) an optimized portion of HBV Core proteinrepresented by SEQ ID NO:99 (positions 483 to 634 of SEQ ID NO:102,e.g., corresponding to positions 37 to 188 of SEQ ID NO:9 plusoptimization of epitopes); (5) an optimized portion of HBV X antigenrepresented by SEQ ID NO:100 (positions 635 to 694 of SEQ ID NO:102,e.g., corresponding to positions 52 to 127 of SEQ ID NO:12 plusoptimization of epitopes); and (6) a hexahistidine tag (e.g., positions695 to 700 of SEQ ID NO:102). In one embodiment, the linker sequence ofthreonine (Thr or T)-serine (Ser or S) is used between the N-terminalpeptide of SEQ ID NO:37 and the first HBV protein (optimized portion ofHBV large surface antigen), thereby extending the total length of SEQ IDNO:102 by two amino acids. In one embodiment, an optimized portion of anHBV large (L) surface antigen used in the fusion protein described aboveis represented by positions 1 to 248 of SEQ ID NO:97 (thereby extendingthe total length of SEQ ID NO:102 by one amino acid). In one embodimentboth the T-S linker and positions 1-248 of SEQ ID NO:97 are used in SEQID NO:102.

As discussed above, the invention includes the modification of HBVantigens from their naturally occurring or wild-type sequences forinclusion in a yeast-based immunotherapeutic that improve the clinicalutility or meet required criteria for therapeutics or prophylacticsrelated to infectious agents, utilizing consensus sequences from a givenHBV genotype to design or form the protein segments. By way of example,additional HBV antigens for use in a yeast-based immunotherapeutic ofthe invention were designed to illustrate this type of modification. Asin the design of the HBV fusion proteins represented described above, toproduce these additional fusion proteins, individual HBV antigens withinthe fusion were optimized or modified to reduce the size of the segmentsin the protein (e.g., to ensure that the protein represented less than ⅔of the HBV genome), as well as to maximize the inclusion of T cellepitopes that have been associated with an immune response inacute/self-limiting HBV infection and/or chronic HBV infection, tomaximize conserved epitopes, to minimize non-natural sequences, and alsoto utilize consensus sequences for each of genotype A-D that were builtfrom multiple sources of HBV sequences (e.g., Yu and Yuan et al, 2010,for S, Core and X, where consensus sequences were generated from 322 HBVsequences, or for Pol (RT), from the Stanford University HIV DrugResistance Database, HBVseq and HBV Site Release Notes). In designingthe following four exemplary fusion proteins comprising HBV antigens,the consensus sequence for the given HBV genotype was used unless usingthe consensus sequence altered one of the known acute self-limiting Tcells epitopes or one of the known polymerase escape mutation sites, inwhich case, these positions followed the published sequence for theseepitopes or mutation sites. Additional antigens could be constructedbased solely on consensus sequences or using other published epitopes asthey become known.

Example 7 describes a fusion protein that is similar in design to thefusion protein represented by SEQ ID NO:101 or SEQ ID NO:102(illustrated schematically by FIG. 3), but that is based on a consensussequence for HBV genotype A. This fusion protein is a single polypeptidewith the following sequence elements fused in frame from N- toC-terminus, represented by SEQ ID NO:107 (optional sequences that arenot HBV sequences are not included in the base sequence of SEQ IDNO:107, but may be added to this sequence as in the construct describedin Example 7): (1) optionally, an N-terminal peptide that is a syntheticN-terminal peptide designed to impart resistance to proteasomaldegradation and stabilize expression represented by SEQ ID NO:37, whichmay be substituted by an N-terminal peptide represented by SEQ ID NO:89,SEQ ID NO:90, or another N-terminal peptide suitable for use with ayeast-based immunotherapeutic as described herein; (2) optionally, alinker peptide of from one to three or more amino acids linker sequencesof one, two, three or more amino acids, such as the two amino acidlinker of Thr-Ser; (3) an optimized portion of an HBV large (L) surfaceantigen represented by positions 1 to 249 of SEQ ID NO:107, which is aconsensus sequence for HBV genotype A utilizing the design strategydiscussed above; (4) an optimized portion of the reverse transcriptase(RT) domain of HBV polymerase represented by positions 250 to 477 of SEQID NO:107, which is a consensus sequence for HBV genotype A utilizingthe design strategy discussed above; (5) an optimized portion of HBVCore protein represented by positions 478 to 629 of SEQ ID NO:107, whichis a consensus sequence for HBV genotype A utilizing the design strategydiscussed above; (6) an optimized portion of HBV X antigen representedby positions 630 to 689 of SEQ ID NO:107, which is a consensus sequencefor HBV genotype A utilizing the design strategy discussed above; and(7) optionally, a hexahistidine tag. A yeast-based immunotherapycomposition expressing this fusion protein is also referred to herein asGI-13010.

Example 7 also describes a fusion protein that is similar in design tothe fusion protein represented by SEQ ID NO:101 or SEQ ID NO:102(illustrated schematically by FIG. 3), but that is based on a consensussequence for HBV genotype B. This fusion protein is a single polypeptidewith the following sequence elements fused in frame from N- toC-terminus, represented by SEQ ID NO:108 (optional sequences that arenot HBV sequences are not included in the base sequence of SEQ IDNO:108, but may be added to this sequence as in the construct describedin Example 7): (1) optionally, an N-terminal peptide that is a syntheticN-terminal peptide designed to impart resistance to proteasomaldegradation and stabilize expression represented by SEQ ID NO:37, whichmay be substituted by an N-terminal peptide represented by SEQ ID NO:89,SEQ ID NO:90, or another N-terminal peptide suitable for use with ayeast-based immunotherapeutic as described herein; (2) optionally, alinker peptide of from one to three or more amino acids linker sequencesof one, two, three or more amino acids, such as the two amino acidlinker of Thr-Ser; (3) an optimized portion of an HBV large (L) surfaceantigen represented by positions 1 to 249 of SEQ ID NO:108, which is aconsensus sequence for HBV genotype B utilizing the design strategydiscussed above; (4) an optimized portion of the reverse transcriptase(RT) domain of HBV polymerase represented by positions 250 to 477 of SEQID NO:108, which is a consensus sequence for HBV genotype B utilizingthe design strategy discussed above; (5) an optimized portion of HBVCore protein represented by positions 478 to 629 of SEQ ID NO:108, whichis a consensus sequence for HBV genotype B utilizing the design strategydiscussed above; (6) an optimized portion of HBV X antigen representedby positions 630 to 689 of SEQ ID NO:108, which is a consensus sequencefor HBV genotype B utilizing the design strategy discussed above; and(7) optionally, a hexahistidine tag. A yeast-based immunotherapycomposition expressing this fusion protein is also referred to herein asGI-13011.

Example 7 also describes a fusion protein that is similar in design tothe fusion protein represented by SEQ ID NO:101 or SEQ ID NO:102(illustrated schematically by FIG. 3), but that is based on a consensussequence for HBV genotype C. This fusion protein is a single polypeptidewith the following sequence elements fused in frame from N- toC-terminus, represented by SEQ ID NO:109 (optional sequences that arenot HBV sequences are not included in the base sequence of SEQ IDNO:109, but may be added to this sequence as in the construct describedin Example 7): (1) optionally, an N-terminal peptide that is a syntheticN-terminal peptide designed to impart resistance to proteasomaldegradation and stabilize expression represented by SEQ ID NO:37, whichmay be substituted by an N-terminal peptide represented by SEQ ID NO:89,SEQ ID NO:90, or another N-terminal peptide suitable for use with ayeast-based immunotherapeutic as described herein; (2) optionally, alinker peptide of from one to three or more amino acids linker sequencesof one, two, three or more amino acids, such as the two amino acidlinker of Thr-Ser; (3) an optimized portion of an HBV large (L) surfaceantigen represented by positions 1 to 249 of SEQ ID NO:109, which is aconsensus sequence for HBV genotype C utilizing the design strategydiscussed above; (4) an optimized portion of the reverse transcriptase(RT) domain of HBV polymerase represented by positions 250 to 477 of SEQID NO:109, which is a consensus sequence for HBV genotype C utilizingthe design strategy discussed above; (5) an optimized portion of HBVCore protein represented by positions 478 to 629 of SEQ ID NO:109, whichis a consensus sequence for HBV genotype C utilizing the design strategydiscussed above; (6) an optimized portion of HBV X antigen representedby positions 630 to 689 of SEQ ID NO:109, which is a consensus sequencefor HBV genotype C utilizing the design strategy discussed above; and(7) optionally, a hexahistidine tag. A yeast-based immunotherapycomposition expressing this fusion protein is also referred to herein asGI-13012.

Example 7 also describes a fusion protein that is similar in design tothe fusion protein represented by SEQ ID NO:101 or SEQ ID NO:102(illustrated schematically by FIG. 3), but that is based on a consensussequence for HBV genotype D. This fusion protein is a single polypeptidewith the following sequence elements fused in frame from N- toC-terminus, represented by SEQ ID NO:110 (optional sequences that arenot HBV sequences are not included in the base sequence of SEQ IDNO:110, but may be added to this sequence as in the construct describedin Example 7): (1) optionally, an N-terminal peptide that is a syntheticN-terminal peptide designed to impart resistance to proteasomaldegradation and stabilize expression represented by SEQ ID NO:37, whichmay be substituted by an N-terminal peptide represented by SEQ ID NO:89,SEQ ID NO:90, or another N-terminal peptide suitable for use with ayeast-based immunotherapeutic as described herein; (2) optionally, alinker peptide of from one to three or more amino acids linker sequencesof one, two, three or more amino acids, such as the two amino acidlinker of Thr-Ser; (3) an optimized portion of an HBV large (L) surfaceantigen represented by positions 1 to 249 of SEQ ID NO:110, which is aconsensus sequence for HBV genotype D utilizing the design strategydiscussed above; (4) an optimized portion of the reverse transcriptase(RT) domain of HBV polymerase represented by positions 250 to 477 of SEQID NO:110, which is a consensus sequence for HBV genotype D utilizingthe design strategy discussed above; (5) an optimized portion of HBVCore protein represented by positions 478 to 629 of SEQ ID NO:110, whichis a consensus sequence for HBV genotype D utilizing the design strategydiscussed above; (6) an optimized portion of HBV X antigen representedby positions 630 to 689 of SEQ ID NO:110, which is a consensus sequencefor HBV genotype D utilizing the design strategy discussed above; and(7) optionally, a hexahistidine tag. A yeast-based immunotherapycomposition expressing this fusion protein which comprises an N-terminalsequence represented by SEQ ID NO:37 is referred to herein as GI-13013.A yeast-based immunotherapy composition expressing this fusion proteinwhich comprises an N-terminal sequence represented by SEQ ID NO:89 isreferred to herein as GI-13014.

As discussed above, it is one embodiment of the invention to change theorder of HBV protein segments within a fusion protein described herein.Accordingly, although the constructs utilizing four HBV proteins asdescribed above are provided in the order of a surface antigen fused toa polymerase antigen fused to a Core antigen fused to an X antigen, theinvention is not limited to this particular order of proteins within theconstruct, and indeed, other arrangements of fusion segments may be usedand in some aspects, may improve the resulting immunotherapeuticcompositions. For example, rearrangement of segments within a fusionprotein may improve or modify expression of the HBV antigen in yeast, ormay improve or modify the immunogenicity or other functional attributeof the HBV antigen. In one aspect of this embodiment, the inventioncontemplates beginning with one HBV antigen that expresses well in yeastand/or provides positive functional data (e.g., is immunogenic), andadding additional HBV proteins or domains to that HBV antigen in orderto expand the potential antigens or epitopes that are contained withinthe HBV antigen. Example 8 provides an example of additionalarrangements of the four HBV proteins described above.

Example 8 describes a fusion protein that contains sequences from HBVsurface antigen, core protein, polymerase and X antigen, where thesequences were derived from segments of the fusion proteins representedby SEQ ID NO:110 and SEQ ID NO:118, and where the fusion proteinutilizes a different order of fusion segments as compared to SEQ IDNO:110. This antigen is based on a consensus sequence for HBV genotypeD; however, it would be straightforward to produce a fusion proteinhaving a similar overall structure using the corresponding fusionsegments from the fusion proteins represented by SEQ ID NO:107 or SEQ IDNO:112 (genotype A), SEQ ID NO:108 or SEQ ID NO:114 (genotype B), SEQ IDNO:109 or SEQ ID NO:116 (genotype C), or using the correspondingsequences from a different HBV genotype, sub-genotype, consensussequence or strain. In this example, yeast (e.g., Saccharomycescerevisiae) were engineered to express this fusion protein under thecontrol of the copper-inducible promoter, CUP1, and the resultingyeast-HBV immunotherapy composition can be referred to herein asGI-13017, schematically illustrated in FIG. 10. The fusion proteinrepresented by SEQ ID NO:124 comprises, in order, surface antigen, core,polymerase and X antigen sequences, as a single polypeptide with thefollowing sequence elements fused in frame from N- to C-terminus,represented by SEQ ID NO:124 (optional sequences that are not HBVsequences are not included in the base sequence of SEQ ID NO:124, butmay be added to this sequence as in the construct described in Example8): (1) optionally, an N-terminal peptide that is a synthetic N-terminalpeptide designed to impart resistance to proteasomal degradation andstabilize expression represented by SEQ ID NO:37 (in the constructdescribed in Example 8), which may be substituted by an N-terminalpeptide represented by SEQ ID NO:89, SEQ ID NO:90, or another N-terminalpeptide suitable for use with a yeast-based immunotherapeutic asdescribed herein; (2) optionally, a linker peptide of from one to threeor more amino acids, such as the two amino acid linker of Thr-Ser (inthe construct described in Example 8); (3) the amino acid sequence of anear full-length (minus position 1) consensus sequence for HBV genotypeD large (L) surface antigen represented by positions 1 to 399 of SEQ IDNO:124 (corresponding to positions 1 to 399 of SEQ ID NO:118); 4) theamino acid sequence of a consensus sequence for HBV genotype D coreantigen represented by positions 400 to 581 of SEQ ID NO:124(corresponding to positions 400 to 581 of SEQ ID NO:118); (5) anoptimized portion of the reverse transcriptase (RT) domain of HBVpolymerase using a consensus sequence for HBV genotype D, represented bypositions 582 to 809 of SEQ ID NO:124 (corresponding to positions to 250to 477 of SEQ ID NO:110); (6) an optimized portion of HBV X antigenusing a consensus sequence for HBV genotype D, represented by positions810 to 869 of SEQ ID NO:124 (corresponding to positions 630 to 689 ofSEQ ID NO:110); and (7) optionally, a hexahistidine tag (in theconstruct described in Example 8). SEQ ID NO:124 contains multiple Tcell epitopes (human and murine), which can be found in Table 5. Anucleic acid sequence encoding the fusion protein of SEQ ID NO:124(codon-optimized for expression in yeast) is represented herein by SEQID NO:123.

Example 8 also describes another fusion protein that contains sequencesfrom HBV surface antigen, core protein, X antigen, and polymerase, wherethe sequences were derived from segments of the fusion proteinsrepresented by SEQ ID NO:110 and SEQ ID NO:118, but where the fusionprotein utilizes a different order of fusion segments as compared to SEQID NO:110. This antigen is also based on a consensus sequence for HBVgenotype D; however, it would be straightforward to produce a fusionprotein having a similar overall structure using the correspondingfusion segments from the fusion proteins represented by SEQ ID NO:107 orSEQ ID NO:112 (genotype A), SEQ ID NO:108 or SEQ ID NO:114 (genotype B),SEQ ID NO:109 or SEQ ID NO:116 (genotype C), or using the correspondingsequences from a different HBV genotype, sub-genotype, consensussequence or strain. In this example, yeast (e.g., Saccharomycescerevisiae) were engineered to express this fusion protein under thecontrol of the copper-inducible promoter, CUP1, and the resultingyeast-HBV immunotherapy composition can be referred to herein asGI-13018, schematically illustrated in FIG. 11. The fusion proteinrepresented by SEQ ID NO:126 comprises, in order, surface antigen, core,X antigen, and polymerase sequences, as a single polypeptide with thefollowing sequence elements fused in frame from N- to C-terminus,represented by SEQ ID NO:126 (optional sequences that are not HBVsequences are not included in the base sequence of SEQ ID NO:126, butmay be added to this sequence as in the construct described in Example8): (1) optionally, an N-terminal peptide that is a synthetic N-terminalpeptide designed to impart resistance to proteasomal degradation andstabilize expression represented by SEQ ID NO:37 (in the constructdescribed in Example 8), which may be substituted by an N-terminalpeptide represented by SEQ ID NO:89, SEQ ID NO:90, or another N-terminalpeptide suitable for use with a yeast-based immunotherapeutic asdescribed herein; (2) optionally, a linker peptide of from one to threeor more amino acids, such as the two amino acid linker of Thr-Ser (inthe construct described in Example 8); (3) the amino acid sequence of anear full-length (minus position 1) consensus sequence for HBV genotypeD large (L) surface antigen represented by positions 1 to 399 of SEQ IDNO:126 (corresponding to positions 1 to 399 of SEQ ID NO:118); 4) theamino acid sequence of a consensus sequence for HBV genotype D coreantigen represented by positions 400 to 581 of SEQ ID NO:126(corresponding to positions 400 to 581 of SEQ ID NO:118); (5) anoptimized portion of HBV X antigen using a consensus sequence for HBVgenotype D, represented by positions 582 to 641 of SEQ ID NO:126(corresponding to positions 630 to 689 of SEQ ID NO:110); (5) anoptimized portion of the reverse transcriptase (RT) domain of HBVpolymerase using a consensus sequence for HBV genotype D, represented bypositions 642 to 869 of SEQ ID NO:126 (corresponding to positions to 250to 477 of SEQ ID NO:110); and (7) optionally, a hexahistidine tag (inthe construct described in Example 8). SEQ ID NO:126 contains multiple Tcell epitopes (human and murine), which can be found in Table 5. Anucleic acid sequence encoding the fusion protein of SEQ ID NO:126(codon-optimized for expression in yeast) is represented herein by SEQID NO:125.

Example 8 describes another fusion protein that contains sequences fromHBV polymerase, X antigen, surface antigen, core protein, where thesequences were derived from segments of the fusion proteins representedby SEQ ID NO:110 and SEQ ID NO:118, but where the fusion proteinutilizes a different order of fusion segments as compared to SEQ IDNO:110. This antigen is based on a consensus sequence for HBV genotypeD; however, it would be straightforward to produce a fusion proteinhaving a similar overall structure using the corresponding fusionsegments from the fusion proteins represented by SEQ ID NO:107 or SEQ IDNO:112 (genotype A), SEQ ID NO:108 or SEQ ID NO:114 (genotype B), SEQ IDNO:109 or SEQ ID NO:116 (genotype C), or using the correspondingsequences from a different HBV genotype, sub-genotype, consensussequence or strain. In this example, yeast (e.g., Saccharomycescerevisiae) were engineered to express this fusion protein under thecontrol of the copper-inducible promoter, CUP1, and the resultingyeast-HBV immunotherapy composition can be referred to herein asGI-13021, schematically illustrated in FIG. 14. The fusion proteinrepresented by SEQ ID NO:132 comprises, in order, polymerase, X antigen,surface antigen, and core, as a single polypeptide with the followingsequence elements fused in frame from N- to C-terminus, represented bySEQ ID NO:132 (optional sequences that are not HBV sequences are notincluded in the base sequence of SEQ ID NO:132, but may be added to thissequence as in the construct described in Example 8): (1) optionally, anN-terminal peptide that is a synthetic N-terminal peptide designed toimpart resistance to proteasomal degradation and stabilize expressionrepresented by SEQ ID NO:37 (in the construct described in Example 8),which may be substituted by an N-terminal peptide represented by SEQ IDNO:89, SEQ ID NO:90, or another N-terminal peptide suitable for use witha yeast-based immunotherapeutic as described herein; (2) optionally, alinker peptide of from one to three or more amino acids, such as the twoamino acid linker of Thr-Ser (in the construct described in Example 8);(3) an optimized portion of the reverse transcriptase (RT) domain of HBVpolymerase using a consensus sequence for HBV genotype D, represented bypositions 1 to 228 of SEQ ID NO:132 (corresponding to positions to 250to 477 of SEQ ID NO:110); (4) an optimized portion of HBV X antigenusing a consensus sequence for HBV genotype D, represented by positions229 to 288 of SEQ ID NO:132 (corresponding to positions 630 to 689 ofSEQ ID NO:110); (5) the amino acid sequence of a near full-length (minusposition 1) consensus sequence for HBV genotype D large (L) surfaceantigen represented by positions 289 to 687 of SEQ ID NO:132(corresponding to positions 1 to 399 of SEQ ID NO:118); (6) the aminoacid sequence of a consensus sequence for HBV genotype D core antigenrepresented by positions 688 to 869 of SEQ ID NO:132 (corresponding topositions 400 to 581 of SEQ ID NO:118); and (7) optionally, ahexahistidine tag (in the construct described in Example 8). SEQ IDNO:132 contains multiple T cell epitopes (human and murine), which canbe found in Table 5. A nucleic acid sequence encoding the fusion proteinof SEQ ID NO:132 (codon-optimized for expression in yeast) isrepresented herein by SEQ ID NO:131.

Example 8 also describes a fusion protein that contains sequences fromHBV X antigen, polymerase, surface antigen, and core protein, where thesequences were derived from segments of the fusion proteins representedby SEQ ID NO:110 and SEQ ID NO:118, but where the fusion proteinutilizes a different order of fusion segments as compared to SEQ IDNO:110. This antigen is based on a consensus sequence for HBV genotypeD; however, it would be straightforward to produce a fusion proteinhaving a similar overall structure using the corresponding fusionsegments from the fusion proteins represented by SEQ ID NO:107 or SEQ IDNO:112 (genotype A), SEQ ID NO:108 or SEQ ID NO:114 (genotype B), SEQ IDNO:109 or SEQ ID NO:116 (genotype C), or using the correspondingsequences from a different HBV genotype, sub-genotype, consensussequence or strain. In this example, yeast (e.g., Saccharomycescerevisiae) were engineered to express this fusion protein under thecontrol of the copper-inducible promoter, CUP1, and the resultingyeast-HBV immunotherapy composition can be referred to herein asGI-13022, schematically illustrated in FIG. 15. The fusion proteinrepresented by SEQ ID NO:134 comprises, in order, X antigen, polymerase,surface antigen, and core protein, as a single polypeptide with thefollowing sequence elements fused in frame from N- to C-terminus,represented by SEQ ID NO:134 (optional sequences that are not HBVsequences are not included in the base sequence of SEQ ID NO:134, butmay be added to this sequence as in the construct described in Example8): (1) optionally, an N-terminal peptide that is a synthetic N-terminalpeptide designed to impart resistance to proteasomal degradation andstabilize expression represented by SEQ ID NO:37 (in the constructdescribed in Example 8), which may be substituted by an N-terminalpeptide represented by SEQ ID NO:89, SEQ ID NO:90, or another N-terminalpeptide suitable for use with a yeast-based immunotherapeutic asdescribed herein; (2) optionally, a linker peptide of from one to threeor more amino acids, such as the two amino acid linker of Thr-Ser (inthe construct described in Example 8); (3) an optimized portion of HBV Xantigen using a consensus sequence for HBV genotype D, represented bypositions 1 to 60 of SEQ ID NO:134 (corresponding to positions 630 to689 of SEQ ID NO:110); (4) an optimized portion of the reversetranscriptase (RT) domain of HBV polymerase using a consensus sequencefor HBV genotype D, represented by positions 61 to 288 of SEQ ID NO:134(corresponding to positions to 250 to 477 of SEQ ID NO:110); (5) theamino acid sequence of a near full-length (minus position 1) consensussequence for HBV genotype D large (L) surface antigen represented bypositions 289 to 687 of SEQ ID NO:134 (corresponding to positions 1 to399 of SEQ ID NO:118); (6) the amino acid sequence of a consensussequence for HBV genotype D core antigen represented by positions 688 to869 of SEQ ID NO:134 (corresponding to positions 400 to 581 of SEQ IDNO:118); and (7) optionally, a hexahistidine tag (in the constructdescribed in Example 8). SEQ ID NO:134 contains multiple T cell epitopes(human and murine), which can be found in Table 5. A nucleic acidsequence encoding the fusion protein of SEQ ID NO:134 (codon-optimizedfor expression in yeast) is represented herein by SEQ ID NO:133.

HBV Antigens Comprising Surface Antigen, Core Protein and X Antigen.

In one embodiment of the invention, the HBV antigen(s) for use in acomposition or method of the invention is a fusion protein comprisingHBV antigens, wherein the HBV antigens comprise or consist of: the HBVsurface antigen (large (L), medium (M) or small (S)) or at least onestructural, functional or immunogenic domain thereof), the HBV coreprotein (HBcAg) or HBV e-antigen (HBeAg) or at least one structural,functional or immunogenic domain thereof, and the HBV X antigen (HBx) orat least one structural, functional or immunogenic domain thereof. Inone aspect, any one or more of the HBV surface antigen, HBV coreprotein, HBV e-antigen, HBV X antigen, or domain thereof, is full-lengthor near full-length. In one aspect, any one or more of the HBV surfaceantigen, HBV core protein, HBV e-antigen, HBV X antigen, or domainthereof comprises at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% of the linear sequence of a full-length HBV surfaceantigen, HBV core protein, HBV e-antigen, HBV X antigen, or domainthereof, respectively, or of the amino acid sequences represented by SEQID NO:97 (optimized HBV surface antigen), SEQ ID NO:99 (optimized coreprotein), SEQ ID NO:100 (optimized X antigen), or a correspondingsequence from another HBV strain, as applicable. In one aspect, any oneor more of the HBV surface antigen, HBV core protein, HBV e-antigen, HBVX antigen, or domain thereof is at least 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identical to a full-length HBV surfaceantigen, HBV core protein, HBV e-antigen, HBV X antigen, or domainthereof, respectively, or to the amino acid sequences represented by SEQID NO:97 (optimized HBV surface antigen), SEQ ID NO:99 (optimized coreprotein), SEQ ID NO:100 (optimized X antigen), or a correspondingsequence from another HBV strain, as applicable. A variety of suitableand exemplary sequences for additional HBV surface antigens, HBV coreantigens, and HBV X antigens useful in this construct are describedherein.

Example 8 describes a fusion protein that contains sequences from HBVsurface antigen, core protein, and X antigen, where the sequences werederived from segments of the fusion proteins represented by SEQ IDNO:110 and SEQ ID NO:118. This antigen is based on a consensus sequencefor HBV genotype D; however, it would be straightforward to produce afusion protein having a similar overall structure using thecorresponding fusion segments from the fusion proteins represented bySEQ ID NO:107 or SEQ ID NO:112 (genotype A), SEQ ID NO:108 or SEQ IDNO:114 (genotype B), SEQ ID NO:109 or SEQ ID NO:116 (genotype C), orusing the corresponding sequences from a different HBV genotype,sub-genotype, consensus sequence or strain. In this example, yeast(e.g., Saccharomyces cerevisiae) were engineered to express this fusionprotein under the control of the copper-inducible promoter, CUP1, andthe resulting yeast-HBV immunotherapy composition can be referred toherein as GI-13016, schematically illustrated in FIG. 9. The fusionprotein represented by SEQ ID NO:122 comprises, in order, surfaceantigen, core, and X antigen sequences, as a single polypeptide with thefollowing sequence elements fused in frame from N- to C-terminus,represented by SEQ ID NO:122 (optional sequences that are not HBVsequences are not included in the base sequence of SEQ ID NO:122, butmay be added to this sequence as in the construct described in Example8): (1) optionally, an N-terminal peptide that is a synthetic N-terminalpeptide designed to impart resistance to proteasomal degradation andstabilize expression represented by SEQ ID NO:37 (in the constructdescribed in Example 8), which may be substituted by an N-terminalpeptide represented by SEQ ID NO:89, SEQ ID NO:90, or another N-terminalpeptide suitable for use with a yeast-based immunotherapeutic asdescribed herein; (2) optionally, a linker peptide of from one to threeor more amino acids, such as the two amino acid linker of Thr-Ser (inthe construct described in Example 8); (3) the amino acid sequence of anear full-length (minus position 1) consensus sequence for HBV genotypeD large (L) surface antigen represented by positions 1 to 399 of SEQ IDNO:122 (corresponding to positions 1 to 399 of SEQ ID NO:118); 4) theamino acid sequence of a consensus sequence for HBV genotype D coreantigen represented by positions 400 to 581 of SEQ ID NO:122(corresponding to positions 400 to 581 of SEQ ID NO:118); (5) anoptimized portion of HBV X antigen using a consensus sequence for HBVgenotype D, represented by positions 582 to 641 of SEQ ID NO:122(corresponding to positions 630 to 689 of SEQ ID NO:110); and (6)optionally, a hexahistidine tag (in the construct described in Example8). SEQ ID NO:122 contains multiple T cell epitopes (human and murine),which can be found in Table 5. A nucleic acid sequence encoding thefusion protein of SEQ ID NO:122 (codon-optimized for expression inyeast) is represented herein by SEQ ID NO:121.

Example 8 also describes a fusion protein that contains sequences fromHBV surface antigen, core protein, and X antigen, where, as in thefusion protein comprising SEQ ID NO:122, the sequences were derived fromsegments of the fusion proteins represented by SEQ ID NO:110 and SEQ IDNO:118. This fusion protein differs from the fusion protein comprisingSEQ ID NO:122, however, in the arrangement of the fusion segments withinthe fusion protein. This antigen is based on a consensus sequence forHBV genotype D; however, it would be straightforward to produce a fusionprotein having a similar overall structure using the correspondingfusion segments from the fusion proteins represented by SEQ ID NO:107 orSEQ ID NO:112 (genotype A), SEQ ID NO:108 or SEQ ID NO:114 (genotype B),SEQ ID NO:109 or SEQ ID NO:116 (genotype C), or using the correspondingsequences from a different HBV genotype, sub-genotype, consensussequence or strain. In this example, yeast (e.g., Saccharomycescerevisiae) were engineered to express this fusion protein under thecontrol of the copper-inducible promoter, CUP1, and the resultingyeast-HBV immunotherapy composition can be referred to herein asGI-13020, schematically illustrated in FIG. 13. The fusion proteinrepresented by SEQ ID NO:130 comprises, in order, X antigen, surfaceantigen, and core antigen sequences, as a single polypeptide with thefollowing sequence elements fused in frame from N- to C-terminus,represented by SEQ ID NO:130 (optional sequences that are not HBVsequences are not included in the base sequence of SEQ ID NO:130, withthe exception of the Leu-Glu linker between the X antigen segment andthe surface antigen segment in the construct exemplified here, but maybe added to this sequence as in the construct described in Example 8):(1) optionally, an N-terminal peptide that is a synthetic N-terminalpeptide designed to impart resistance to proteasomal degradation andstabilize expression represented by SEQ ID NO:37 (in the constructdescribed in Example 8), which may be substituted by an N-terminalpeptide represented by SEQ ID NO:89, SEQ ID NO:90, or another N-terminalpeptide suitable for use with a yeast-based immunotherapeutic asdescribed herein; (2) optionally, a linker peptide of from one to threeor more amino acids, such as the two amino acid linker of Thr-Ser (inthe construct described in Example 8); (3) an optimized portion of HBV Xantigen using a consensus sequence for HBV genotype D, represented bypositions 1 to 60 of SEQ ID NO:130 (corresponding to positions 630 to689 of SEQ ID NO:110); (4) optionally, a linker peptide of from one tothree or more amino acids, such as the two amino acid linker of Leu-Glu(in the construct described in Example 8), represented by positions 61to 62 of SEQ ID NO:130; (5) the amino acid sequence of a nearfull-length (minus position 1) consensus sequence for HBV genotype Dlarge (L) surface antigen represented by positions 63 to 461 of SEQ IDNO:130 (corresponding to positions 1 to 399 of SEQ ID NO:118); (6) theamino acid sequence of a consensus sequence for HBV genotype D coreantigen represented by positions 462 to 643 of SEQ ID NO:130(corresponding to positions 400 to 581 of SEQ ID NO:118); and (7)optionally, a hexahistidine tag (in the construct described in Example8). SEQ ID NO:130 contains multiple T cell epitopes (human and murine),which can be found in Table 5. The amino acid sequence of the completefusion protein described in Example 8 comprising SEQ ID NO:130 andincluding the N- and C-terminal peptides and all linkers is representedherein by SEQ ID NO:150. A nucleic acid sequence encoding the fusionprotein of SEQ ID NO:130 or SEQ ID NO:150 (codon-optimized forexpression in yeast) is represented herein by SEQ ID NO:129.

HBV Antigens Comprising Surface Antigen, Core Protein and Polymerase.

In one embodiment of the invention, the HBV antigen(s) for use in acomposition or method of the invention is a fusion protein comprisingHBV antigens, wherein the HBV antigens comprise or consist of: the HBVsurface antigen (large (L), medium (M) or small (S)) or at least onestructural, functional or immunogenic domain thereof), the HBV coreprotein (HBcAg) or HBV e-antigen (HBeAg) or at least one structural,functional or immunogenic domain thereof, and the HBV polymerase or atleast one structural, functional or immunogenic domain thereof (e.g.,the reverse transcriptase (RT) domain). In one aspect, any one or moreof the HBV surface antigen, HBV core protein, HBV e-antigen, HBVpolymerase, or domain thereof, is full-length or near full-length. Inone aspect, any one or more of the HBV surface antigen, HBV coreprotein, HBV e-antigen, HBV polymerase, or domain thereof comprises atleast 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% ofthe linear sequence of a full-length HBV surface antigen, HBV coreprotein, HBV e-antigen, HBV polymerase, or domain thereof, respectively,or of the amino acid sequences represented by SEQ ID NO:97 (optimizedHBV surface antigen), SEQ ID NO:99 (optimized core protein), SEQ IDNO:98 (optimized polymerase), or a corresponding sequence from anotherHBV strain, as applicable. In one aspect, any one or more of the HBVsurface antigen, HBV core protein, HBV e-antigen, HBV polymerase, ordomain thereof is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identical to a full-length HBV surface antigen, HBVcore protein, HBV e-antigen, HBV polymerase, or domain thereof,respectively, or to the amino acid sequences represented by SEQ ID NO:97(optimized HBV surface antigen), SEQ ID NO:99 (optimized core protein),SEQ ID NO:98 (optimized polymerase), or a corresponding sequence fromanother HBV strain, as applicable. A variety of suitable and exemplarysequences for HBV surface antigens, HBV polymerase antigens, and HBVcore antigens are described herein.

One example of such a fusion protein is schematically represented inFIG. 7. An example of a composition comprising this fusion protein isdescribed in Example 3. In this embodiment, yeast (e.g., Saccharomycescerevisiae) are engineered to express various HBVsurface-polymerase-core fusion proteins under the control of thecopper-inducible promoter, CUP1, or the TEF2 promoter. In each case, thefusion protein is a single polypeptide with the following sequenceelements fused in frame from N- to C-terminus, represented by SEQ IDNO:41: (1) an N-terminal peptide to impart resistance to proteasomaldegradation and stabilize expression (e.g., positions 1 to 5 of SEQ IDNO:41); 2) an amino acid sequence of the amino HBV hepatocyte receptordomain of the pre-S1 portion of HBV large (L) surface protein (unique toL) (e.g., positions 21-47 of SEQ ID NO:11 or positions 6 to 32 of SEQ IDNO:41); 3) the amino acid sequence of an HBV small (S) surface protein(e.g., positions 176 to 400 of SEQ ID NO:11 or positions 33 to 257 ofSEQ ID NO:41); 4) a two amino acid spacer/linker to facilitate cloningand manipulation of the sequences (e.g., positions 258 and 259 of SEQ IDNO:41); 5) the amino acid sequence of an HBV polymerase comprising thereverse transcriptase domain (e.g., positions 247 to 691 of SEQ ID NO:10or positions 260 to 604 of SEQ ID NO:41); 6) the amino acid sequence ofan HBV core protein (e.g., positions 31-212 of SEQ ID NO:9 or positions605 to 786 of SEQ ID NO:41); and 7) a hexahistidine tag (e.g., positions787 to 792 of SEQ ID NO:41). The sequence also contains epitopes ordomains that are believed to enhance the immunogenicity of the fusionprotein. In addition, in one embodiment, the sequence of this constructcan be modified to introduce one or more or all of the followinganti-viral resistance mutations: rtM2041, rtL180M, rtM204V, rtV173L,rtN236T, rtA194T (positions given with respect to the full-length aminoacid sequence for HBV polymerase). In one embodiment, six differentimmunotherapy compositions are created, each one containing one of thesemutations. In other embodiments, all or some of the mutations areincluded in a single fusion protein. In one embodiment, this constructalso contains one or more anti-viral resistance mutations in the surfaceantigen. The amino acid segments used in any of the fusion proteinsdescribed herein can be modified by the use of additional amino acidsflanking either end of any domain; the examples provided herein areexemplary. For example, a fusion protein according to this embodimentcan include 1) an amino acid sequence of the amino HBV hepatocytereceptor domain of the pre-S1 portion of HBV large (L) surface protein(unique to L) (e.g., positions 21-47 of SEQ ID NO:11 or positions 6 to32 of SEQ ID NO:41); 2) the amino acid sequence of an HBV small (S)surface protein (e.g., positions 176 to 400 of SEQ ID NO:11 or positions33 to 257 of SEQ ID NO:41); 3) the amino acid sequence of an HBVpolymerase comprising the reverse transcriptase domain (e.g., positions247 to 691 of SEQ ID NO:10 or positions 260 to 604 of SEQ ID NO:41); and4) the amino acid sequence of an HBV core protein (e.g., positions31-212 of SEQ ID NO:9 or positions 605 to 786 of SEQ ID NO:41), andutilize no N- or C-terminal sequences, or utilize different N- orC-terminal sequences, and/or use linkers or no linkers between HBVsequences. In one embodiment, instead of the N-terminal peptiderepresented by positions 1-5 of SEQ ID NO:41, an N-terminal peptiderepresented by SEQ ID NO:89 or SEQ ID NO:90 is utilized, followed by theremainder of the fusion protein as described.

Another example of such a fusion protein is described in Example 8.Example 8 exemplifies a fusion protein that contains sequences from HBVsurface antigen, core protein, and polymerase where the sequences werederived from segments of the fusion proteins represented by SEQ IDNO:110 and SEQ ID NO:118. This antigen is based on a consensus sequencefor HBV genotype D; however, it would be straightforward to produce afusion protein having a similar overall structure using thecorresponding fusion segments from the fusion proteins represented bySEQ ID NO:107 or SEQ ID NO:112 (genotype A), SEQ ID NO:108 or SEQ IDNO:114 (genotype B), SEQ ID NO:109 or SEQ ID NO:116 (genotype C), orusing the corresponding sequences from a different HBV genotype,sub-genotype, consensus sequence or strain. In this example, yeast(e.g., Saccharomyces cerevisiae) were engineered to express this fusionprotein under the control of the copper-inducible promoter, CUP1, andthe resulting yeast-HBV immunotherapy composition can be referred toherein as GI-13015, schematically illustrated in FIG. 8. The fusionprotein represented by SEQ ID NO:120 comprises, in order, surfaceantigen, core protein, and polymerase sequences, as a single polypeptidewith the following sequence elements fused in frame from N- toC-terminus, represented by SEQ ID NO:120 (optional sequences that arenot HBV sequences are not included in the base sequence of SEQ IDNO:120, but may be added to this sequence as in the construct describedin Example 8): (1) optionally, an N-terminal peptide that is a syntheticN-terminal peptide designed to impart resistance to proteasomaldegradation and stabilize expression represented by SEQ ID NO:37 (in theconstruct described in Example 8), which may be substituted by anN-terminal peptide represented by SEQ ID NO:89, SEQ ID NO:90, or anotherN-terminal peptide suitable for use with a yeast-based immunotherapeuticas described herein; (2) optionally, a linker peptide of from one tothree or more amino acids, such as the two amino acid linker of Thr-Ser(in the construct described in Example 8); (3) the amino acid sequenceof a near full-length (minus position 1) consensus sequence for HBVgenotype D large (L) surface antigen represented by positions 1 to 399of SEQ ID NO:120 (corresponding to positions 1 to 399 of SEQ ID NO:118);(4) the amino acid sequence of a consensus sequence for HBV genotype Dcore antigen represented by positions 400 to 581 of SEQ ID NO:120(corresponding to positions 400 to 581 of SEQ ID NO:118); (5) anoptimized portion of the reverse transcriptase (RT) domain of HBVpolymerase using a consensus sequence for HBV genotype D, represented bypositions 582 to 809 of SEQ ID NO:120 (corresponding to positions to 250to 477 of SEQ ID NO:110); and (6) optionally, a hexahistidine tag (inthe construct described in Example 8). SEQ ID NO:120 contains multiple Tcell epitopes (human and murine), which can be found in Table 5. Anucleic acid sequence encoding the fusion protein of SEQ ID NO:120(codon-optimized for expression in yeast) is represented herein by SEQID NO:119.

Yet another example of such a fusion protein is described in Example 8.Example 8 exemplifies a fusion protein that contains sequences from HBVpolymerase, surface antigen, and core protein, where the sequences werederived from segments of the fusion proteins represented by SEQ IDNO:110 and SEQ ID NO:118. This fusion protein differs from the fusionprotein comprising SEQ ID NO:120 in the arrangement of the fusionsegments within the fusion protein. This antigen is based on a consensussequence for HBV genotype D; however, it would be straightforward toproduce a fusion protein having a similar overall structure using thecorresponding fusion segments from the fusion proteins represented bySEQ ID NO:107 or SEQ ID NO:112 (genotype A), SEQ ID NO:108 or SEQ IDNO:114 (genotype B), SEQ ID NO:109 or SEQ ID NO:116 (genotype C), orusing the corresponding sequences from a different HBV genotype,sub-genotype, consensus sequence or strain. In this example, yeast(e.g., Saccharomyces cerevisiae) were engineered to express this fusionprotein under the control of the copper-inducible promoter, CUP1, andthe resulting yeast-HBV immunotherapy composition can be referred toherein as GI-13019, schematically illustrated in FIG. 12. The fusionprotein represented by SEQ ID NO:128 comprises, in order, polymerase,surface antigen, and core sequences, as a single polypeptide with thefollowing sequence elements fused in frame from N- to C-terminus,represented by SEQ ID NO:128 (optional sequences that are not HBVsequences are not included in the base sequence of SEQ ID NO:128, withthe exception of the Leu-Glu linker between the polymerase segment andthe surface antigen segment in the construct exemplified here, but maybe added to this sequence as in the construct described in Example 8):(1) optionally, an N-terminal peptide that is a synthetic N-terminalpeptide designed to impart resistance to proteasomal degradation andstabilize expression represented by SEQ ID NO:37 (in the constructdescribed in Example 8), which may be substituted by an N-terminalpeptide represented by SEQ ID NO:89, SEQ ID NO:90, or another N-terminalpeptide suitable for use with a yeast-based immunotherapeutic asdescribed herein; (2) optionally, a linker peptide of from one to threeor more amino acids, such as the two amino acid linker of Thr-Ser (inthe construct described in Example 8); (3) an optimized portion of thereverse transcriptase (RT) domain of HBV polymerase using a consensussequence for HBV genotype D, represented by positions 1 to 228 of SEQ IDNO:128 (corresponding to positions to 250 to 477 of SEQ ID NO:110); (4)optionally, a linker peptide of from one to three or more amino acids,such as the two amino acid linker of Leu-Glu (in the construct describedin Example 8), represented by positions 229 to 230 of SEQ ID NO:128; (5)the amino acid sequence of a near full-length (minus position 1)consensus sequence for HBV genotype D large (L) surface antigenrepresented by positions 231 to 629 of SEQ ID NO:128 (corresponding topositions 1 to 399 of SEQ ID NO:118); (6) the amino acid sequence of aconsensus sequence for HBV genotype D core antigen represented bypositions 630 to 811 of SEQ ID NO:128 (corresponding to positions 400 to581 of SEQ ID NO:118); and (7) optionally, a hexahistidine tag (in theconstruct described in Example 8). SEQ ID NO:128 contains multiple Tcell epitopes (human and murine), which can be found in Table 5. Anucleic acid sequence encoding the fusion protein of SEQ ID NO:128(codon-optimized for expression in yeast) is represented herein by SEQID NO:127.

HBV Antigens Comprising Polymerase and Core Protein.

In one embodiment of the invention, the HBV antigen(s) for use in acomposition or method of the invention is a fusion protein comprisingHBV antigens, wherein the HBV antigens comprise or consist of HBVpolymerase (the RT domain) or at least one immunogenic domain thereofand an HBV core protein (HBcAg) or at least one immunogenic domainthereof. In one aspect, one or both of the RT domain of HBV polymeraseor the HBV core protein is full-length or near full-length. In oneaspect, one or both of the RT domain of HBV polymerase or the HBV coreprotein or a domain thereof comprises at least 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% of the linear sequence of afull-length the RT domain of HBV polymerase or the HBV core protein or adomain thereof, respectively, or to the amino acid sequences representedby SEQ ID NO:98 (optimized HBV polymerase), SEQ ID NO:99 (optimized coreprotein), or a corresponding sequence from another HBV strain, asapplicable. In one aspect, one or both of the RT domain of HBVpolymerase or the HBV core protein or a domain thereof is at least 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to afull-length RT domain of HBV polymerase or the HBV core protein or adomain thereof, respectively, or to the amino acid sequences representedby SEQ ID NO:98 (optimized HBV polymerase), SEQ ID NO:99 (optimized coreprotein), or a corresponding sequence from another HBV strain, asapplicable. A variety of suitable and exemplary sequences for HBVpolymerase antigens and HBV core antigens are described herein.

One example of this antigen is schematically represented in FIG. 4. Oneexample of a composition comprising this fusion protein is described inExample 3. In this embodiment, yeast (e.g., Saccharomyces cerevisiae)are engineered to express various HBV polymerase-core fusion proteins asshown schematically in FIG. 4 under the control of the copper-induciblepromoter, CUP1, or the TEF2 promoter. In each case, the fusion proteinis a single polypeptide with the following sequence elements fused inframe from N- to C-terminus, represented by SEQ ID NO:38: (1) anN-terminal peptide to impart resistance to proteasomal degradation andstabilize expression (e.g., SEQ ID NO:37 or positions 1 to 6 of SEQ IDNO:38); 2) the amino acid sequence of a portion of the HBV genotype Cpolymerase including the reverse transcriptase domain (e.g., positions347 to 691 of SEQ ID NO:10 or positions 7 to 351 of SEQ ID NO:38); 3) anHBV genotype C core protein (e.g., positions 31 to 212 of SEQ ID NO:9 orpositions 352 to 533 of SEQ ID NO:38); and 4) a hexahistidine tag (e.g.,positions 534 to 539 of SEQ ID NO:38). The sequence also containsepitopes or domains that are believed to enhance the immunogenicity ofthe fusion protein. The amino acid segments used in any of the fusionproteins described herein can be modified by the use of additional aminoacids flanking either end of any domain; the examples provided hereinare exemplary. For example, a fusion protein according to thisembodiment can include 1) the amino acid sequence of a portion of theHBV genotype C polymerase including the reverse transcriptase domain(e.g., positions 347 to 691 of SEQ ID NO:10 or positions 7 to 351 of SEQID NO:38); and 2) an HBV genotype C core protein (e.g., positions 31 to212 of SEQ ID NO:9 or positions 352 to 533 of SEQ ID NO:38), and utilizeno N- or C-terminal sequences, or utilize different N- or C-terminalsequences, and/or use linkers or no linkers between HBV sequences. Inone embodiment, instead of the N-terminal peptide represented by SEQ IDNO:37, an N-terminal peptide represented by SEQ ID NO:89 or SEQ ID NO:90is utilized, followed by the remainder of the fusion protein.

HBV Antigens Comprising X Antigen and Core Protein.

In one embodiment of the invention, the HBV antigen(s) for use in acomposition or method of the invention is a fusion protein comprisingHBV antigens, wherein the HBV antigens comprise or consist of HBV Xantigen or at least one immunogenic domain thereof and HBV core protein(HBcAg) or at least one immunogenic domain thereof. In one aspect, oneor both of the HBV X antigen or the HBV core protein is full-length ornear full-length. In one aspect, one or both of the HBV X antigen or theHBV core protein or a domain thereof comprises at least 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the linear sequence ofa full-length HBV X antigen or HBV core protein or domain thereof,respectively, or to the amino acid sequences represented by SEQ ID NO:99(optimized core protein), SEQ ID NO:100 (optimized X antigen), or acorresponding sequence from another HBV strain, as applicable. In oneaspect, one or both of the HBV X antigen or the HBV core protein or adomain thereof is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identical to a full-length HBV X antigen or HBV coreprotein or domain thereof, respectively, or to the amino acid sequencesrepresented by SEQ ID NO:99 (optimized core protein), SEQ ID NO:100(optimized X antigen), or a corresponding sequence from another HBVstrain, as applicable. A variety of suitable and exemplary sequences forHBV core antigens and HBV X antigens are described herein.

This fusion protein is schematically represented in FIG. 5. An exampleof a composition comprising this fusion protein is described in Example3. In this embodiment, yeast (e.g., Saccharomyces cerevisiae) areengineered to express various HBV X-core fusion proteins as shownschematically in FIG. 5 under the control of the copper-induciblepromoter, CUP1, or the TEF2 promoter. In each case, the fusion proteinis a single polypeptide with the following sequence elements fused inframe from N- to C-terminus, represented by SEQ ID NO:39 (1) anN-terminal peptide to impart resistance to proteasomal degradation andstabilize expression (e.g. SEQ ID NO:37 or positions 1 to 6 of SEQ IDNO:39); 2) the amino acid sequence of a near full-length (minusposition 1) HBV genotype C X antigen (e.g., positions 2 to 154 of SEQ IDNO:12 or positions 7 to 159 of SEQ ID NO:39); 3) an HBV genotype C coreprotein (e.g., positions 31 to 212 of SEQ ID NO:9 or positions 160 to341 of SEQ ID NO:39); and 4) a hexahistidine tag (e.g., positions 342 to347 of SEQ ID NO:39). The sequence also contains epitopes or domainsthat are believed to enhance the immunogenicity of the fusion protein.The amino acid segments used in any of the fusion proteins describedherein can be modified by the use of additional amino acids flankingeither end of any domain; the examples provided herein are exemplary.For example, a fusion protein according to this embodiment caninclude 1) the amino acid sequence of a near full-length (minusposition 1) HBV genotype C X antigen (e.g., positions 2 to 154 of SEQ IDNO:12 or positions 7 to 159 of SEQ ID NO:39); and 2) an HBV genotype Ccore protein (e.g., positions 31 to 212 of SEQ ID NO:9 or positions 160to 341 of SEQ ID NO:39), and utilize no N- or C-terminal sequences, orutilize different N- or C-terminal sequences, and/or use linkers or nolinkers between HBV sequences. In one embodiment, instead of theN-terminal peptide represented by SEQ ID NO:37, an N-terminal peptiderepresented by SEQ ID NO:89 or SEQ ID NO:90 is utilized, followed by theremainder of the fusion protein as described.

HBV Antigens Comprising Single HBV Proteins.

In one embodiment of the invention, an HBV antigen is comprised of asingle HBV protein (e.g., one HBV protein selected from surface, core,e-antigen, polymerase, or X antigen) or one or more domains (structural,functional, and/or immunological) from a single HBV protein. Thisembodiment of the invention is particularly useful for creating ayeast-based immunotherapeutic composition that can be used, for example,in combination with one or more other yeast-based immunotherapeuticcompositions for the treatment or prophylaxis of HBV, or in sequencewith one or more other yeast-based immunotherapeutic compositions forthe treatment or prophylaxis of HBV, or to follow a prophylacticapproach with a therapeutic approach if the patient becomes infected.For example, the yeast-based immunotherapeutic composition including anHBV surface antigen of this embodiment can be combined with a secondyeast-based immunotherapeutic composition including a different HBVprotein/antigen, such as an HBV X antigen (described below), andfurther, with additional “single HBV protein” yeast-basedimmunotherapeutics, as desired (e.g., a yeast-based immunotherapeuticcomposition including an HBV Precore, Core or e-antigen and/or ayeast-based immunotherapeutic composition including an HBV polymeraseantigen or domain thereof). These “single HBV protein yeastimmunotherapeutics” can be used in combination or sequence with eachother and/or in combination or sequence with other multi-HBV proteinyeast-based immunotherapeutics, such as those described in the Examplesor elsewhere herein. Alternatively, or in addition, a “single HBVprotein yeast immunotherapeutic” such as this HBV surface antigenyeast-based immunotherapeutic can be produced using the HBV sequence forany given genotype or sub-genotype, and additional HBV surface antigenyeast-based immunotherapeutics can be produced using the HBV sequencesfor any one or more additional genotype or sub-genotype. This strategyeffectively creates a “spice rack” of different HBV antigens andgenotypes and/or sub-genotypes to each of which is provided in thecontext of a yeast-based immunotherapeutic of the invention, or in astrategy that includes at least one yeast-based immunotherapeutic of theinvention. Accordingly, any combination of one, two, three, four, five,six, seven, eight, nine, ten or more of these yeast-basedimmunotherapeutics can be selected for use to treat a particular patientor population of patients who are infected with HBV, illustrating theflexibility of the present invention to be customized or tailored tomeet the needs of a particular patient, population of patients,demographic, or other patient grouping.

In one embodiment of the invention, the HBV antigen(s) for use in acomposition or method of the invention is an HBV antigen comprising orconsisting of: (a) an HBV surface antigen protein and/or one or moredomains (structural, functional or immunogenic) thereof, which caninclude the hepatocyte receptor portion of Pre-S1 of the HBV large (L)surface antigen, the HBV large (L) surface antigen, the HBV middle (M)surface antigen, the HBV small (S) surface antigen (HBsAg), or anydomain or combination thereof; (b) an HBV polymerase antigen, which caninclude one or more domains (structural, functional, or immunogenic) ofHBV polymerase, such as the reverse transcriptase (RT) domain (afunctional domain) of HBV polymerase; (c) an HBV precore antigen, an HBVcore antigen and/or HBV e-antigen, or one or more domains thereof(structural, functional or immunogenic), which can include one or moredomains or portions of HBV Precore containing sequences from both HBVcore and HBV e-antigen, or one or the other of these proteins; or (d) anHBV X antigen, which can include one or more domains (structural,functional or immunogenic) of HBV X antigen. In one aspect, any one ormore of these proteins or domains is full-length or near full-length. Inone aspect, one or more of these proteins or domains comprise or consistof 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more immunogenic domains. In oneaspect, any one or more of these proteins or domains comprises at least80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99° A of thelinear sequence of the corresponding full-length sequence or a domainthereof. In one aspect, any one or more of these proteins or domains isat least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to the corresponding full-length sequence or a domain thereof.A variety of suitable and exemplary sequences for HBV surface antigens,HBV polymerase antigens, HBV core antigens, and HBV X antigens aredescribed herein.

An example of a composition comprising a surface antigen protein isdescribed in Example 5. In this embodiment, yeast (e.g., Saccharomycescerevisiae) are engineered to express HBV surface proteins under thecontrol of a suitable promoter, such as the copper-inducible promoter,CUP1, or the TEF2 promoter. The protein is a single polypeptidecomprising HBV near-full-length HBV large (L) surface antigen (toaccommodate the presence of an N-terminal sequence selected to enhanceor stabilize expression of the antigen), represented by SEQ ID NO:93:(1) an N-terminal peptide of SEQ ID NO:89 (positions 1-89 of SEQ IDNO:93); 2) the amino acid sequence of a near full-length (minusposition 1) HBV genotype C large (L) surface antigen (e.g., positions2-400 of SEQ ID NO:11 or positions 90 to 488 of SEQ ID NO:93); and 3) ahexahistidine tag (e.g., positions 489 to 494 of SEQ ID NO:93).Alternatively, the N-terminal peptide can be replaced with SEQ ID NO:37or a homologue thereof or another N-terminal peptide described herein.In one embodiment, this construct also contains one or more anti-viralresistance mutations in the surface antigen. While this example utilizeslarge (L) surface antigen as an HBV antigen that may maximize theexposure of immunogenic epitopes generated by the immune system, smallportions of surface antigen, including any domains or combinations ofdomains of surface antigen, can be produced using the guidance providedherein. In addition, while the exemplary immunotherapeutic is shownusing a genotype C sequence, sequences from other genotypes,sub-genotypes, and/or strains or isolates of HBV can be used instead.

An example of a composition comprising an HBV polymerase antigen isdescribed in Example 3 and also in Example 5. The HBV antigen describedin Example 5 is schematically represented in FIG. 6. In this embodiment,yeast (e.g., Saccharomyces cerevisiae) are engineered to express variousHBV polymerase proteins under the control of the copper-induciblepromoter, CUP1, or the TEF2 promoter. In each case, the fusion proteinis a single polypeptide with the following sequence elements fused inframe from N- to C-terminus, represented by SEQ ID NO:40 (1) anN-terminal peptide to impart resistance to proteasomal degradation andstabilize expression (SEQ ID NO:37, or positions 1 to 6 of SEQ ID NO:40;2) the amino acid sequence of a portion of the HBV genotype C polymeraseincluding the reverse transcriptase domain (e.g., positions 347 to 691of SEQ ID NO:10 or positions 7 to 351 of SEQ ID NO:40); and 3) ahexahistidine tag (e.g., positions 352 to 357 of SEQ ID NO:40). Thesequence also contains epitopes or domains that are believed to enhancethe immunogenicity of the fusion protein. In addition, in oneembodiment, the sequence of this construct can be modified to introduceone or more or all of the following anti-viral resistance mutations:rtM2041, rtL180M, rtM204V, rtV173L, rtN236T, rtA194T (positions givenwith respect to the full-length amino acid sequence for HBV polymerase).In one embodiment, six different immunotherapy compositions are created,each one containing one of these mutations. In other embodiments, all orsome of the mutations are included in a single fusion protein. The aminoacid segments used in any of the fusion proteins described herein can bemodified by the use of additional amino acids flanking either end of anydomain; the examples provided herein are exemplary. For example, afusion protein according to this embodiment can include the amino acidsequence of a portion of the HBV genotype C polymerase including thereverse transcriptase domain (e.g., positions 347 to 691 of SEQ ID NO:10or positions 7 to 351 of SEQ ID NO:40), and utilize no N- or C-terminalsequences, or utilize different N- or C-terminal sequences, and/or uselinkers or no linkers between HBV sequences. In one embodiment, insteadof the N-terminal peptide represented by SEQ ID NO:37, an N-terminalpeptide represented by SEQ ID NO:89 or SEQ ID NO:90 is utilized,followed by the remainder of the fusion protein as described.

In the embodiment shown in Example 5, yeast (e.g., Saccharomycescerevisiae) are engineered to express HBV polymerase proteins under thecontrol of a suitable promoter, such as the copper-inducible promoter,CUP1, or the TEF2 promoter. The protein is a single polypeptidecomprising HBV reverse transcriptase (RT) domain of polymerase (Pol),represented by SEQ ID NO:94: (1) an N-terminal peptide of SEQ ID NO:89(positions 1-89 of SEQ ID NO:94); 2) the amino acid sequence of aportion of the HBV genotype C polymerase including the reversetranscriptase domain (e.g., positions 347 to 691 of SEQ ID NO:10 orpositions 90 to 434 of SEQ ID NO:94); and 3) a hexahistidine tag (e.g.,positions 435 to 440 of SEQ ID NO:94). The sequence also containsepitopes or domains that are believed to enhance the immunogenicity ofthe fusion protein. In addition, in one embodiment, the sequence of thisconstruct can be modified to introduce one or more or all of thefollowing anti-viral resistance mutations: rtM2041, rtL180M, rtM204V,rtV173L, rtN236T, rtA194T (positions given with respect to thefull-length amino acid sequence for HBV polymerase). Alternatively, theN-terminal peptide can be replaced with SEQ ID NO:37 or a homologuethereof or another N-terminal peptide described herein.

An example of a composition comprising an HBV Precore, Core or e-antigenis described in Example 5. Yeast (e.g., Saccharomyces cerevisiae) areengineered to express HBV Core proteins under the control of a suitablepromoter, such as the copper-inducible promoter, CUP1, or the TEF2promoter. The protein is a single polypeptide comprising nearfull-length HBV Core protein, represented by SEQ ID NO:95: (1) anN-terminal peptide of SEQ ID NO:89 (positions 1-89 of SEQ ID NO:95); 2)the amino acid sequence of a portion of the HBV genotype C Core protein(e.g., positions 31 to 212 of SEQ ID NO:9 or positions 90 to 271 of SEQID NO:95); and 3) a hexahistidine tag (e.g., positions 272 to 277 of SEQID NO:95). The sequence also contains epitopes or domains that arebelieved to enhance the immunogenicity of the fusion protein.Alternatively, the N-terminal peptide can be replaced with SEQ ID NO:37or a homologue thereof or another N-terminal peptide described herein.

An example of a yeast-based immunotherapeutic composition comprising anHBV X antigen is described in Example 5. Yeast (e.g., Saccharomycescerevisiae) are engineered to express HBV X antigens under the controlof a suitable promoter, such as the copper-inducible promoter, CUP1, orthe TEF2 promoter. The protein is a single polypeptide comprising nearfull-length HBV X antigen, represented by SEQ ID NO:96: (1) anN-terminal peptide of SEQ ID NO:89 (positions 1-89 of SEQ ID NO:96); 2)the amino acid sequence of a portion of the HBV genotype C X antigen(e.g., positions 2 to 154 of SEQ ID NO:12 or positions 90 to 242 of SEQID NO:96); and 3) a hexahistidine tag (e.g., positions 243 to 248 of SEQID NO:96). The sequence also contains epitopes or domains that arebelieved to enhance the immunogenicity of the fusion protein.Alternatively, the N-terminal peptide can be replaced with SEQ ID NO:37or a homologue thereof or another N-terminal peptide described herein.

HBV Antigens Comprising HBV Proteins from Two or More Genotypes.

Another embodiment of the invention relates to HBV antigens for use inan immunotherapeutic composition of the invention that maximizes thetargeting of HBV genotypes and/or sub-genotypes in order to providecompositions with the potential to treat a large number of individualsor populations of individuals using one composition. Such compositionsare generally more efficient to produce (i.e., have a productionadvantage by including multiple antigens and/or a consensus approach totargeting genotypes) and are more efficient to utilize in a wide varietyof clinical settings (e.g., one composition may serve many differenttypes of patient populations in many different geographical settings).As discussed above, to produce such HBV antigens, conserved antigensand/or conserved domains (among HBV genotypes) can be selected, and theantigens can be designed to maximize the inclusion of conservedimmunological domains.

In one aspect of this embodiment, an HBV antigen is provided thatincludes in a single yeast-based immunotherapeutic a single HBV proteinor domain thereof (e.g., surface, polymerase, core/e or X) that isrepeated two, three, four, five or more times within the antigenconstruct, each time using a sequence from a different HBV genotype orsubgenotype. In this aspect, multiple dominant or prevalent genotypescan be targeted in one yeast-based immunotherapeutic, increasingclinical and manufacturing efficacy. These antigens can be modified, ifdesired, to maximize the inclusion of consensus sequences, includingconsensus T cell epitopes within the antigens, which may otherwisecontain subtle differences due to sub-genotype, strain or isolatedifferences.

Accordingly, in one embodiment of the invention, the HBV antigen(s) foruse in a composition or method of the invention is an HBV antigencomprising or consisting of two or more repeated HBV antigens of thesame protein or domain, but of different HBV genotypes (e.g., two ormore HBV Core or e-antigens, which can include one or more domains(structural, functional or immunogenic) of HBV Core or e-antigen,wherein the antigens include the same or similar antigen from each ofHBV genotype C and HBV genotype D, to form a Core-Core fusion where eachCore protein is a different genotype). In one aspect, the HBV proteinused in such constructs is full-length or near full-length protein ordomain. In one aspect, the HBV antigen comprises or consists of 1, 2, 3,4, 5, 6, 7, 8, 9, or 10 or more immunogenic domains. In one aspect, anyone or more of these proteins or domains comprises at least 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the linearsequence of the corresponding full-length sequence. In one aspect, anyone or more of these proteins or domains is at least 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence ofthe corresponding full-length sequence.

Such an antigen is exemplified in Example 6. In this embodiment, yeast(e.g., Saccharomyces cerevisiae) are engineered to express an HBV fusionprotein under the control of a suitable promoter, such as thecopper-inducible promoter, CUP1, or the TEF2 promoter. The protein is asingle polypeptide comprising four Core antigens, each one from adifferent genotype (HBV genotypes A, B, C and D), represented by SEQ IDNO:105: 1) an N-terminal methionine at position 1 of SEQ ID NO:105; 2)the amino acid sequence of a near full-length Core protein from HBVgenotype A (e.g., positions 31 to 212 of SEQ ID NO:1 or positions 2 to183 of SEQ ID NO: 105); 3) the amino acid sequence of a near full-lengthCore protein from HBV genotype B (e.g., positions 30 to 212 of SEQ IDNO:5 or positions 184 to 395 of SEQ ID NO: 105); 4) the amino acidsequence of a near full-length Core protein from HBV genotype C (e.g.,positions 30 to 212 of SEQ ID NO:9 or positions 396 to 578 of SEQ ID NO:105); 5) the amino acid sequence of a near full-length Core protein fromHBV genotype D (e.g., positions 30 to 212 of SEQ ID NO:13 or positions579 to 761 of SEQ ID NO: 105); and 5) a hexahistidine tag (e.g.,positions 762 to 767 of SEQ ID NO: 105). The sequence also containsepitopes or domains that are believed to enhance the immunogenicity ofthe fusion protein. The N-terminal methionine at position 1 can besubstituted with SEQ ID NO:37 or a homologue thereof, or with an alphaprepro sequence of SEQ ID NO:89 or SEQ ID NO:90, or a homologue thereof,or any other suitable N-terminal sequence if desired. In addition,linker sequences can be inserted between HBV proteins to facilitatecloning and manipulation of the construct, if desired. This is anexemplary construct, as any other combination of HBV genotypes and/orsub-genotypes can be substituted into this design as desired toconstruct a single antigen yeast-based HBV immunotherapeutic productwith broad clinical applicability and efficient design formanufacturing. The amino acid sequence of SEQ ID NO:105 also containsseveral known T cell epitopes, and certain epitopes have been modifiedto correspond to the published sequence for the given epitope (see Table5).

In another aspect of this embodiment, more than one protein or domainfrom a single HBV genotype is included in an HBV antigen useful in theinvention, which may be selected to maximize the most conserved proteinsequences encoded by the HBV genome or to maximize the inclusion oftherapeutically or prophylactically useful immunogenic domains withinthe antigen. These antigens are then repeated within the same fusionprotein, but using the same or similar sequences from a different HBVgenotype or subgenotype. In this aspect, multiple dominant or prevalentgenotypes can also be targeted in one yeast-based immunotherapeutic,again increasing clinical and manufacturing efficacy. These antigens canalso be modified, if desired, to maximize the inclusion of consensus Tcell epitopes within the antigens, which may otherwise contain subtledifferences due to sub-genotype, strain or isolate differences.

Accordingly, in one embodiment of the invention, the HBV antigen(s) foruse in a composition or method of the invention is an HBV antigencomprising or consisting of at least two different HBV proteins ordomains thereof, each of which is repeated two or more times, butwherein the repeated sequences are from different HBV genotypes (e.g.,two or more HBV Core and two or more X antigens, or domains thereof,wherein the antigens include the same or similar antigen from each ofHBV genotype C and HBV genotype D, to form a Core-X-Core-X fusion (orany other order of segments within the fusion) where each Core proteinis a different genotype and each X antigen is a different genotype). Inone aspect, the HBV protein used in such constructs is full-length ornear full-length protein or domain. In one aspect, the HBV antigencomprises or consists of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or moreimmunogenic domains. In one aspect, any one or more of these proteins ordomains comprises at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% of the linear sequence of the corresponding full-lengthsequence. In one aspect, any one or more of these proteins or domains isat least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%to the sequence of the corresponding full-length sequence.

Such an antigen is exemplified in Example 6. In this embodiment, yeast(e.g., Saccharomyces cerevisiae) are engineered to express an HBV fusionprotein under the control of a suitable promoter, such as thecopper-inducible promoter, CUP1, or the TEF2 promoter. The protein is asingle polypeptide comprising two Core antigens and two X antigens, eachone of the pair from a different genotype (HBV genotypes A and C),represented by SEQ ID NO:106: 1) an N-terminal methionine at position 1of SEQ ID NO:106; 2) the amino acid sequence of a near full-length Coreprotein from HBV genotype A (e.g., positions 31 to 212 of SEQ ID NO:1 orpositions 2 to 183 of SEQ ID NO:106); 3) the amino acid sequence of afull-length X antigen from HBV genotype A (e.g., positions SEQ ID NO:4or positions 184 to 337 of SEQ ID NO:106); 4) the amino acid sequence ofa near full-length Core protein from HBV genotype C (e.g., positions 30to 212 of SEQ ID NO:9 or positions 338 to 520 of SEQ ID NO:106); 5) theamino acid sequence of a full-length X antigen from HBV genotype C(e.g., SEQ ID NO:8 or positions 521 to 674 of SEQ ID NO:106); and 5) ahexahistidine tag (e.g., positions 675 to 680 of SEQ ID NO:106). Thesequence also contains epitopes or domains that are believed to enhancethe immunogenicity of the fusion protein. The N-terminal methionine atposition 1 can be substituted with SEQ ID NO:37 or a homologue thereof,or with an alpha prepro sequence of SEQ ID NO:89 or SEQ ID NO:90, or ahomologue thereof. The amino acid sequence of SEQ ID NO:106 alsocontains several known T cell epitopes, and certain epitopes have beenmodified to correspond to the published sequence for the given epitope(see Table 5).

Additional Embodiments Regarding HBV Antigens.

In some aspects of the invention, amino acid insertions, deletions,and/or substitutions can be made for one, two, three, four, five, six,seven, eight, nine, ten, or more amino acids of a wild-type or referenceHBV protein, provided that the resulting HBV protein, when used as anantigen in a yeast-HBV immunotherapeutic composition of the invention,elicits an immune response against the target or wild-type or referenceHBV protein, which may include an enhanced immune response, a diminishedimmune response, or a substantially similar immune response. Forexample, the invention includes the use of HBV agonist antigens, whichmay include one or more T cell epitopes that have been mutated toenhance the T cell response against the HBV agonist, such as byimproving the avidity or affinity of the epitope for an MHC molecule orfor the T cell receptor that recognizes the epitope in the context ofMHC presentation. HBV protein agonists may therefore improve the potencyor efficiency of a T cell response against native HBV proteins thatinfect a host.

Referring to any of the above-described HBV antigens, including thefusion proteins that have amino acid sequences including or representedby SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40,SEQ ID NO:41, SEQ ID NO:92, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:107,SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:112, SEQ IDNO:114, SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:122, SEQID NO:124, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:130, SEQ ID NO:132,SEQ ID NO:134, SEQ ID NO:150 or SEQ ID NO:151, it is an aspect of theinvention to use one or more of the HBV antigens from individual HBVproteins within the fusion protein (e.g., from HBV surface antigen, HBVpolymerase, HBV core/e-antigen, and/or HBV X antigen) to construct“single protein” antigens (e.g., antigens from only one of these HBVproteins), or to construct fusion proteins using only two or three ofthe HBV protein segments, if applicable to the given reference fusionprotein. It is also an aspect of the invention to change the order ofHBV protein segments within the fusion protein. As another alternatedesign, HBV genotypes and/or consensus sequences can be combined, wheretwo, three, four or more genotypes and/or consensus sequences are usedto construct the fusion protein.

The invention also includes homologues of any of the above-describedfusion proteins, as well as the use of homologues, variants, or mutantsof the individual HBV proteins or portions thereof (including anyfunctional and/or immunogenic domains) that are part of such fusionproteins or otherwise described herein. In one aspect, the inventionincludes the use of fusion proteins or individual (single) HBV proteinsor HBV antigens, having amino acid sequences that are at least 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to the amino acid sequence of any one of the fusion proteinsor individual HBV proteins or HBV antigens, respectively, describedherein, including any of the HBV proteins, HBV antigens and fusionproteins referenced by a specific sequence identifier herein, over thefull length of the fusion protein, or with respect to a defined segmentin the fusion protein or a defined protein or domain thereof(immunogenic domain or functional domain (i.e., a domain with at leastone biological activity)) that forms part of the fusion protein. ManyCTL epitopes (epitopes that are recognized by cytotoxic T lymphocytesfrom patients infected with HBV) and escape mutations (mutations thatarise in an HBV protein due to selective pressure from an anti-viraldrug) are known in the art, and this information can also be used tomake substitutions or create variants or homologues of the HBV antigensdescribed herein in order to provide a specific sequence in the HBVantigen of the invention.

Yeast-Based Immunotherapy Compositions.

In various embodiments of the invention, the invention includes the useof at least one “yeast-based immunotherapeutic composition” (whichphrase may be used interchangeably with “yeast-based immunotherapyproduct”, “yeast-based immunotherapy composition”, “yeast-basedcomposition”, “yeast-based immunotherapeutic”, “yeast-based vaccine”, orderivatives of these phrases). An “immunotherapeutic composition” is acomposition that elicits an immune response sufficient to achieve atleast one therapeutic benefit in a subject. As used herein, yeast-basedimmunotherapeutic composition refers to a composition that includes ayeast vehicle component and that elicits an immune response sufficientto achieve at least one therapeutic benefit in a subject. Moreparticularly, a yeast-based immunotherapeutic composition is acomposition that includes a yeast vehicle component and can elicit orinduce an immune response, such as a cellular immune response, includingwithout limitation a T cell-mediated cellular immune response. In oneaspect, a yeast-based immunotherapeutic composition useful in theinvention is capable of inducing a CD8⁺ and/or a CD4⁺ T cell-mediatedimmune response and in one aspect, a CD8⁺ and a CD4⁺ T cell-mediatedimmune response. Optionally, a yeast-based immunotherapeutic compositionis capable of eliciting a humoral immune response. A yeast-basedimmunotherapeutic composition useful in the present invention can, forexample, elicit an immune response in an individual such that theindividual is protected from HBV infection and/or is treated for HBVinfection or for symptoms resulting from HBV infection.

Yeast-based immunotherapy compositions of the invention may be either“prophylactic” or “therapeutic”. When provided prophylactically, thecompositions of the present invention are provided in advance of anysymptom of HBV infection. Such a composition could be administered atbirth, in early childhood, or to adults. The prophylactic administrationof the immunotherapy compositions serves to prevent subsequent HBVinfection, to resolve an infection more quickly or more completely ifHBV infection subsequently ensues, and/or to ameliorate the symptoms ofHBV infection if infection subsequently ensues. When providedtherapeutically, the immunotherapy compositions are provided at or afterthe onset of HBV infection, with the goal of ameliorating at least onesymptom of the infection and preferably, with a goal of eliminating theinfection, providing a long lasting remission of infection, and/orproviding long term immunity against subsequent infections orreactivations of the virus. In one aspect, a goal of treatment is lossof detectable HBV viral load or reduction of HBV viral load (e.g., belowdetectable levels by PCR or <2000 IU/ml). In one aspect, a goal oftreatment is sustained viral clearance for at least 6 months after thecompletion of therapy. In one aspect, a goal of treatment is the loss ofdetectable serum HBeAg and/or HBsAg proteins. In one aspect, a goal oftreatment is the development of antibodies against the hepatitis Bsurface antigen (anti-HBs) and/or antibodies against HBeAg. In oneaspect, the goal of treatment is seroconversion, which may be definedby: (a) 10 or more sample ratio units (SRU) as determined byradioimmunoassay; (b) a positive result as determined by enzymeimmunoassay; or (c) detection of an antibody concentration of ≧10 mIU/ml(10 SRU is comparable to 10 mIU/mL of antibody). In one aspect, a goalof treatment is normalization of serum alanine aminotransferase (ALT)levels, improvement in liver inflammation and/or improvement in liverfibrosis.

Typically, a yeast-based immunotherapy composition includes a yeastvehicle and at least one antigen or immunogenic domain thereof expressedby, attached to, or mixed with the yeast vehicle, wherein the antigen isheterologous to the yeast, and wherein the antigen comprises one or moreHBV antigens or immunogenic domains thereof. In some embodiments, theantigen or immunogenic domain thereof is provided as a fusion protein.Several HBV fusion proteins suitable for use in the compositions andmethods of the invention have been described above. In one aspect of theinvention, fusion protein can include two or more antigens. In oneaspect, the fusion protein can include two or more immunogenic domainsof one or more antigens, or two or more epitopes of one or moreantigens.

In any of the yeast-based immunotherapy compositions used in the presentinvention, the following aspects related to the yeast vehicle areincluded in the invention. According to the present invention, a yeastvehicle is any yeast cell (e.g., a whole or intact cell) or a derivativethereof (see below) that can be used in conjunction with one or moreantigens, immunogenic domains thereof or epitopes thereof in atherapeutic composition of the invention, or in one aspect, the yeastvehicle can be used alone or as an adjuvant. The yeast vehicle cantherefore include, but is not limited to, a live intact (whole) yeastmicroorganism (i.e., a yeast cell having all its components including acell wall), a killed (dead) or inactivated intact yeast microorganism,or derivatives of intact/whole yeast including: a yeast spheroplast(i.e., a yeast cell lacking a cell wall), a yeast cytoplast (i.e., ayeast cell lacking a cell wall and nucleus), a yeast ghost (i.e., ayeast cell lacking a cell wall, nucleus and cytoplasm), a subcellularyeast membrane extract or fraction thereof (also referred to as a yeastmembrane particle and previously as a subcellular yeast particle), anyother yeast particle, or a yeast cell wall preparation.

Yeast spheroplasts are typically produced by enzymatic digestion of theyeast cell wall. Such a method is described, for example, in Franzusoffet al., 1991, Meth. Enzymol. 194, 662-674, incorporated herein byreference in its entirety.

Yeast cytoplasts are typically produced by enucleation of yeast cells.Such a method is described, for example, in Coon, 1978, Natl. CancerInst. Monogr. 48, 45-55 incorporated herein by reference in itsentirety.

Yeast ghosts are typically produced by resealing a permeabilized orlysed cell and can, but need not, contain at least some of theorganelles of that cell. Such a method is described, for example, inFranzusoff et al., 1983, J. Biol. Chem. 258, 3608-3614 and Bussey etal., 1979, Biochim. Biophys. Acta 553, 185-196, each of which isincorporated herein by reference in its entirety.

A yeast membrane particle (subcellular yeast membrane extract orfraction thereof) refers to a yeast membrane that lacks a naturalnucleus or cytoplasm. The particle can be of any size, including sizesranging from the size of a natural yeast membrane to microparticlesproduced by sonication or other membrane disruption methods known tothose skilled in the art, followed by resealing. A method for producingsubcellular yeast membrane extracts is described, for example, inFranzusoff et al., 1991, Meth. Enzymol. 194, 662-674. One may also usefractions of yeast membrane particles that contain yeast membraneportions and, when the antigen or other protein was expressedrecombinantly by the yeast prior to preparation of the yeast membraneparticles, the antigen or other protein of interest. Antigens or otherproteins of interest can be carried inside the membrane, on eithersurface of the membrane, or combinations thereof (i.e., the protein canbe both inside and outside the membrane and/or spanning the membrane ofthe yeast membrane particle). In one embodiment, a yeast membraneparticle is a recombinant yeast membrane particle that can be an intact,disrupted, or disrupted and resealed yeast membrane that includes atleast one desired antigen or other protein of interest on the surface ofthe membrane or at least partially embedded within the membrane.

An example of a yeast cell wall preparation is a preparation of isolatedyeast cell walls carrying an antigen on its surface or at leastpartially embedded within the cell wall such that the yeast cell wallpreparation, when administered to an animal, stimulates a desired immuneresponse against a disease target.

Any yeast strain can be used to produce a yeast vehicle of the presentinvention. Yeast are unicellular microorganisms that belong to one ofthree classes: Ascomycetes, Basidiomycetes and Fungi Imperfecti. Oneconsideration for the selection of a type of yeast for use as an immunemodulator is the pathogenicity of the yeast. In one embodiment, theyeast is a non-pathogenic strain such as Saccharomyces cerevisiae. Theselection of a non-pathogenic yeast strain minimizes any adverse effectsto the individual to whom the yeast vehicle is administered. However,pathogenic yeast may be used if the pathogenicity of the yeast can benegated by any means known to one of skill in the art (e.g., mutantstrains).

Genera of yeast strains that may be used in the invention include butare not limited to Saccharomyces, Candida, Cryptococcus, Hansenula,Kluyveromyces, Pichia, Rhodotorula, Schizosaccharomyces and Yarrowia. Inone aspect, yeast genera are selected from Saccharomyces, Candida,Hansenula, Pichia or Schizosaccharomyces, and in one aspect, yeastgenera are selected from Saccharomyces, Hansenula, and Pichia, and inone aspect, Saccharomyces is used. Species of yeast strains that may beused in the invention include but are not limited to Saccharomycescerevisiae, Saccharomyces carlsbergensis, Candida albicans, Candidakefyr, Candida tropicalis, Cryptococcus laurentii, Cryptococcusneoformans, Hansenula anomala, Hansenula polymorpha, Kluyveromycesfragilis, Kluyveromyces lactis, Kluyveromyces marxianus var. lactis,Pichia pastoris, Rhodotorula rubra, Schizosaccharomyces pombe, andYarrowia lipolytica. It is to be appreciated that a number of thesespecies include a variety of subspecies, types, subtypes, etc. that areintended to be included within the aforementioned species. In oneaspect, yeast species used in the invention include S. cerevisiae, C.albicans, H. polymorpha, P. pastoris and S. pombe. S. cerevisiae isuseful as it is relatively easy to manipulate and being “GenerallyRecognized As Safe” or “GRAS” for use as food additives (GRAS, FDAproposed Rule 62FR18938, Apr. 17, 1997). One embodiment of the presentinvention is a yeast strain that is capable of replicating plasmids to aparticularly high copy number, such as a S. cerevisiae cir° strain. TheS. cerevisiae strain is one such strain that is capable of supportingexpression vectors that allow one or more target antigen(s) and/orantigen fusion protein(s) and/or other proteins to be expressed at highlevels. In addition, any mutant yeast strains can be used in the presentinvention, including those that exhibit reduced post-translationalmodifications of expressed target antigens or other proteins, such asmutations in the enzymes that extend N-linked glycosylation.

In most embodiments of the invention, the yeast-based immunotherapycomposition includes at least one antigen, immunogenic domain thereof,or epitope thereof. The antigens contemplated for use in this inventioninclude any HBV antigen or immunogenic domain thereof, includingmutants, variants and agonists of HBV proteins or domains thereof,against which it is desired to elicit an immune response for the purposeof prophylactically or therapeutically immunizing a host against HBVinfection. HBV antigens that are useful in various embodiments of theinvention have been described in detail above.

Optionally, proteins, including fusion proteins, which are used as acomponent of the yeast-based immunotherapeutic composition of theinvention are produced using constructs that are particularly useful forimproving or enhancing the expression, or the stability of expression,of recombinant antigens in yeast. Typically, the desired antigenicprotein(s) or peptide(s) are fused at their amino-terminal end to: (a) aspecific synthetic peptide that stabilizes the expression of the fusionprotein in the yeast vehicle or prevents posttranslational modificationof the expressed fusion protein (such peptides are described in detail,for example, in U.S. Patent Publication No. 2004-0156858 A1, publishedAug. 12, 2004, incorporated herein by reference in its entirety); (b) atleast a portion of an endogenous yeast protein, including but notlimited to alpha factor, wherein either fusion partner provides improvedstability of expression of the protein in the yeast and/or a preventspost-translational modification of the proteins by the yeast cells (suchproteins are also described in detail, for example, in U.S. PatentPublication No. 2004-0156858 A1, supra); and/or (c) at least a portionof a yeast protein that causes the fusion protein to be expressed on thesurface of the yeast (e.g., an Aga protein, described in more detailherein). In addition, the present invention optionally includes the useof peptides that are fused to the C-terminus of the antigen-encodingconstruct, particularly for use in the selection and identification ofthe protein. Such peptides include, but are not limited to, anysynthetic or natural peptide, such as a peptide tag (e.g.,hexahistidine) or any other short epitope tag. Peptides attached to theC-terminus of an antigen according to the invention can be used with orwithout the addition of the N-terminal peptides discussed above.

In one embodiment, a synthetic peptide useful in a fusion protein islinked to the N-terminus of the antigen, the peptide consisting of atleast two amino acid residues that are heterologous to the antigen,wherein the peptide stabilizes the expression of the fusion protein inthe yeast vehicle or prevents posttranslational modification of theexpressed fusion protein. The synthetic peptide and N-terminal portionof the antigen together form a fusion protein that has the followingrequirements: (1) the amino acid residue at position one of the fusionprotein is a methionine (i.e., the first amino acid in the syntheticpeptide is a methionine); (2) the amino acid residue at position two ofthe fusion protein is not a glycine or a proline (i.e., the second aminoacid in the synthetic peptide is not a glycine or a proline); (3) noneof the amino acid residues at positions 2-6 of the fusion protein is amethionine (i.e., the amino acids at positions 2-6, whether part of thesynthetic peptide or the protein, if the synthetic peptide is shorterthan 6 amino acids, do not include a methionine); and (4) none of theamino acids at positions 2-6 of the fusion protein is a lysine or anarginine (i.e., the amino acids at positions 2-6, whether part of thesynthetic peptide or the protein, if the synthetic peptide is shorterthan 5 amino acids, do not include a lysine or an arginine). Thesynthetic peptide can be as short as two amino acids, but in one aspect,is 2-6 amino acids (including 3, 4, 5 amino acids), and can be longerthan 6 amino acids, in whole integers, up to about 200 amino acids, 300amino acids, 400 amino acids, 500 amino acids, or more.

In one embodiment, a fusion protein comprises an amino acid sequence ofM-X2-X3-X4-X5-X6, wherein M is methionine; wherein X2 is any amino acidexcept glycine, proline, lysine or arginine; wherein X3 is any aminoacid except methionine, lysine or arginine; wherein X4 is any amino acidexcept methionine, lysine or arginine; wherein X5 is any amino acidexcept methionine, lysine or arginine; and wherein X6 is any amino acidexcept methionine, lysine or arginine. In one embodiment, the X6 residueis a proline. An exemplary synthetic sequence that enhances thestability of expression of an antigen in a yeast cell and/or preventspost-translational modification of the protein in the yeast includes thesequence M-A-D-E-A-P (SEQ ID NO:37). Another exemplary syntheticsequence with the same properties is M-V. In addition to the enhancedstability of the expression product, these fusion partners do not appearto negatively impact the immune response against the immunizing antigenin the construct. In addition, the synthetic fusion peptides can bedesigned to provide an epitope that can be recognized by a selectionagent, such as an antibody.

In one embodiment, the HBV antigen is linked at the N-terminus to ayeast protein, such as an alpha factor prepro sequence (also referred toas the alpha factor signal leader sequence, the amino acid sequence ofwhich is exemplified herein by SEQ ID NO:89 or SEQ ID NO:90. Othersequences for yeast alpha factor prepro sequence are known in the artand are encompassed for use in the present invention.

In one aspect of the invention, the yeast vehicle is manipulated suchthat the antigen is expressed or provided by delivery or translocationof an expressed protein product, partially or wholly, on the surface ofthe yeast vehicle (extracellular expression). One method foraccomplishing this aspect of the invention is to use a spacer arm forpositioning one or more protein(s) on the surface of the yeast vehicle.For example, one can use a spacer arm to create a fusion protein of theantigen(s) or other protein of interest with a protein that targets theantigen(s) or other protein of interest to the yeast cell wall. Forexample, one such protein that can be used to target other proteins is ayeast protein (e.g., cell wall protein 2 (cwp2), Aga2, Pir4 or Flo1protein) that enables the antigen(s) or other protein to be targeted tothe yeast cell wall such that the antigen or other protein is located onthe surface of the yeast. Proteins other than yeast proteins may be usedfor the spacer arm; however, for any spacer arm protein, it is mostdesirable to have the immunogenic response be directed against thetarget antigen rather than the spacer arm protein. As such, if otherproteins are used for the spacer arm, then the spacer arm protein thatis used should not generate such a large immune response to the spacerarm protein itself such that the immune response to the targetantigen(s) is overwhelmed. One of skill in the art should aim for asmall immune response to the spacer arm protein relative to the immuneresponse for the target antigen(s). Spacer arms can be constructed tohave cleavage sites (e.g., protease cleavage sites) that allow theantigen to be readily removed or processed away from the yeast, ifdesired. Any known method of determining the magnitude of immuneresponses can be used (e.g., antibody production, lytic assays, etc.)and are readily known to one of skill in the art.

Another method for positioning the target antigen(s) or other proteinsto be exposed on the yeast surface is to use signal sequences such asglycosylphosphatidyl inositol (GPI) to anchor the target to the yeastcell wall. Alternatively, positioning can be accomplished by appendingsignal sequences that target the antigen(s) or other proteins ofinterest into the secretory pathway via translocation into theendoplasmic reticulum (ER) such that the antigen binds to a proteinwhich is bound to the cell wall (e.g., cwp).

In one aspect, the spacer arm protein is a yeast protein. The yeastprotein can consist of between about two and about 800 amino acids of ayeast protein. In one embodiment, the yeast protein is about 10 to 700amino acids. In another embodiment, the yeast protein is about 40 to 600amino acids. Other embodiments of the invention include the yeastprotein being at least 250 amino acids, at least 300 amino acids, atleast 350 amino acids, at least 400 amino acids, at least 450 aminoacids, at least 500 amino acids, at least 550 amino acids, at least 600amino acids, or at least 650 amino acids. In one embodiment, the yeastprotein is at least 450 amino acids in length. Another consideration foroptimizing antigen surface expression, if that is desired, is whetherthe antigen and spacer arm combination should be expressed as a monomeror as dimer or as a trimer, or even more units connected together. Thisuse of monomers, dimers, trimers, etc. allows for appropriate spacing orfolding of the antigen such that some part, if not all, of the antigenis displayed on the surface of the yeast vehicle in a manner that makesit more immunogenic.

Use of yeast proteins can stabilize the expression of fusion proteins inthe yeast vehicle, prevents posttranslational modification of theexpressed fusion protein, and/or targets the fusion protein to aparticular compartment in the yeast (e.g., to be expressed on the yeastcell surface). For delivery into the yeast secretory pathway, exemplaryyeast proteins to use include, but are not limited to: Aga (including,but not limited to, Aga1 and/or Aga2); SUC2 (yeast invertase); alphafactor signal leader sequence; CPY; Cwp2p for its localization andretention in the cell wall; BUD genes for localization at the yeast cellbud during the initial phase of daughter cell formation; Flo1p; Pir2p;and Pir4p.

Other sequences can be used to target, retain and/or stabilize theprotein to other parts of the yeast vehicle, for example, in the cytosolor the mitochondria or the endoplasmic reticulum or the nucleus.Examples of suitable yeast protein that can be used for any of theembodiments above include, but are not limited to, TK, AF, SEC7;phosphoenolpyruvate carboxykinase PCK1, phosphoglycerokinase PGK andtriose phosphate isomerase TPI gene products for their repressibleexpression in glucose and cytosolic localization; the heat shockproteins SSA1, SSA3, SSA4, SSC1, whose expression is induced and whoseproteins are more thermostable upon exposure of cells to heat treatment;the mitochondrial protein CYC1 for import into mitochondria; ACT 1.

Methods of producing yeast vehicles and expressing, combining and/orassociating yeast vehicles with antigens and/or other proteins and/oragents of interest to produce yeast-based immunotherapy compositions arecontemplated by the invention.

According to the present invention, the term “yeast vehicle-antigencomplex” or “yeast-antigen complex” is used generically to describe anyassociation of a yeast vehicle with an antigen, and can be usedinterchangeably with “yeast-based immunotherapy composition” when suchcomposition is used to elicit an immune response as described above.Such association includes expression of the antigen by the yeast (arecombinant yeast), introduction of an antigen into a yeast, physicalattachment of the antigen to the yeast, and mixing of the yeast andantigen together, such as in a buffer or other solution or formulation.These types of complexes are described in detail below.

In one embodiment, a yeast cell used to prepare the yeast vehicle istransfected with a heterologous nucleic acid molecule encoding a protein(e.g., the antigen) such that the protein is expressed by the yeastcell. Such a yeast is also referred to herein as a recombinant yeast ora recombinant yeast vehicle. The yeast cell can then be loaded into thedendritic cell as an intact cell, or the yeast cell can be killed, or itcan be derivatized such as by formation of yeast spheroplasts,cytoplasts, ghosts, or subcellular particles, any of which is followedby loading of the derivative into the dendritic cell. Yeast spheroplastscan also be directly transfected with a recombinant nucleic acidmolecule (e.g., the spheroplast is produced from a whole yeast, and thentransfected) in order to produce a recombinant spheroplast thatexpresses an antigen or other protein.

In general, the yeast vehicle and antigen(s) and/or other agents can beassociated by any technique described herein. In one aspect, the yeastvehicle was loaded intracellularly with the antigen(s) and/or agent(s).In another aspect, the antigen(s) and/or agent(s) was covalently ornon-covalently attached to the yeast vehicle. In yet another aspect, theyeast vehicle and the antigen(s) and/or agent(s) were associated bymixing. In another aspect, and in one embodiment, the antigen(s) and/oragent(s) is expressed recombinantly by the yeast vehicle or by the yeastcell or yeast spheroplast from which the yeast vehicle was derived.

A number of antigens and/or other proteins to be produced by a yeastvehicle of the present invention is any number of antigens and/or otherproteins that can be reasonably produced by a yeast vehicle, andtypically ranges from at least one to at least about 6 or more,including from about 2 to about 6 heterologous antigens and or otherproteins.

Expression of an antigen or other protein in a yeast vehicle of thepresent invention is accomplished using techniques known to thoseskilled in the art. Briefly, a nucleic acid molecule encoding at leastone desired antigen or other protein is inserted into an expressionvector in such a manner that the nucleic acid molecule is operativelylinked to a transcription control sequence in order to be capable ofeffecting either constitutive or regulated expression of the nucleicacid molecule when transformed into a host yeast cell. Nucleic acidmolecules encoding one or more antigens and/or other proteins can be onone or more expression vectors operatively linked to one or moreexpression control sequences. Particularly important expression controlsequences are those which control transcription initiation, such aspromoter and upstream activation sequences. Any suitable yeast promotercan be used in the present invention and a variety of such promoters areknown to those skilled in the art. Promoters for expression inSaccharomyces cerevisiae include, but are not limited to, promoters ofgenes encoding the following yeast proteins: alcohol dehydrogenase I(ADH1) or II (ADH2), CUP1, phosphoglycerate kinase (PGK), triosephosphate isomerase (TPI), translational elongation factor EF-1 alpha(TEF2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; also referredto as TDH3, for triose phosphate dehydrogenase), galactokinase (GAL1),galactose-1-phosphate uridyl-transferase (GAL7), UDP-galactose epimerase(GAL10), cytochrome c1 (CYC1), Sec7 protein (SEC7) and acid phosphatase(PHO5), including hybrid promoters such as ADH2/GAPDH and CYC1/GAL10promoters, and including the ADH2/GAPDH promoter, which is induced whenglucose concentrations in the cell are low (e.g., about 0.1 to about 0.2percent), as well as the CUP1 promoter and the TEF2 promoter. Likewise,a number of upstream activation sequences (UASs), also referred to asenhancers, are known. Upstream activation sequences for expression inSaccharomyces cerevisiae include, but are not limited to, the UASs ofgenes encoding the following proteins: PCK1, TPI, TDH3, CYC1, ADH1,ADH2, SUC2, GAL1, GAL7 and GAL10, as well as other UASs activated by theGAL4 gene product, with the ADH2 UAS being used in one aspect. Since theADH2 UAS is activated by the ADR1 gene product, it may be preferable tooverexpress the ADR1 gene when a heterologous gene is operatively linkedto the ADH2 UAS. Transcription termination sequences for expression inSaccharomyces cerevisiae include the termination sequences of theα-factor, GAPDH, and CYC1 genes.

Transcription control sequences to express genes in methyltrophic yeastinclude the transcription control regions of the genes encoding alcoholoxidase and formate dehydrogenase.

Transfection of a nucleic acid molecule into a yeast cell according tothe present invention can be accomplished by any method by which anucleic acid molecule can be introduced into the cell and includes, butis not limited to, diffusion, active transport, bath sonication,electroporation, microinjection, lipofection, adsorption, and protoplastfusion. Transfected nucleic acid molecules can be integrated into ayeast chromosome or maintained on extrachromosomal vectors usingtechniques known to those skilled in the art. Examples of yeast vehiclescarrying such nucleic acid molecules are disclosed in detail herein. Asdiscussed above, yeast cytoplast, yeast ghost, and yeast membraneparticles or cell wall preparations can also be produced recombinantlyby transfecting intact yeast microorganisms or yeast spheroplasts withdesired nucleic acid molecules, producing the antigen therein, and thenfurther manipulating the microorganisms or spheroplasts using techniquesknown to those skilled in the art to produce cytoplast, ghost orsubcellular yeast membrane extract or fractions thereof containingdesired antigens or other proteins.

Effective conditions for the production of recombinant yeast vehiclesand expression of the antigen and/or other protein by the yeast vehicleinclude an effective medium in which a yeast strain can be cultured. Aneffective medium is typically an aqueous medium comprising assimilablecarbohydrate, nitrogen and phosphate sources, as well as appropriatesalts, minerals, metals and other nutrients, such as vitamins and growthfactors. The medium may comprise complex nutrients or may be a definedminimal medium. Yeast strains of the present invention can be culturedin a variety of containers, including, but not limited to, bioreactors,Erlenmeyer flasks, test tubes, microtiter dishes, and Petri plates.Culturing is carried out at a temperature, pH and oxygen contentappropriate for the yeast strain. Such culturing conditions are wellwithin the expertise of one of ordinary skill in the art (see, forexample, Guthrie et al. (eds.), 1991, Methods in Enzymology, vol. 194,Academic Press, San Diego).

In some embodiments of the invention, yeast are grown under neutral pHconditions. As used herein, the general use of the term “neutral pH”refers to a pH range between about pH 5.5 and about pH 8, and in oneaspect, between about pH 6 and about 8. One of skill the art willappreciate that minor fluctuations (e.g., tenths or hundredths) canoccur when measuring with a pH meter. As such, the use of neutral pH togrow yeast cells means that the yeast cells are grown in neutral pH forthe majority of the time that they are in culture. In one embodiment,yeast are grown in a medium maintained at a pH level of at least 5.5(i.e., the pH of the culture medium is not allowed to drop below pH5.5). In another aspect, yeast are grown at a pH level maintained atabout 6, 6.5, 7, 7.5 or 8. The use of a neutral pH in culturing yeastpromotes several biological effects that are desirable characteristicsfor using the yeast as vehicles for immunomodulation. For example,culturing the yeast in neutral pH allows for good growth of the yeastwithout negative effect on the cell generation time (e.g., slowing ofdoubling time). The yeast can continue to grow to high densities withoutlosing their cell wall pliability. The use of a neutral pH allows forthe production of yeast with pliable cell walls and/or yeast that aremore sensitive to cell wall digesting enzymes (e.g., glucanase) at allharvest densities. This trait is desirable because yeast with flexiblecell walls can induce different or improved immune responses as comparedto yeast grown under more acidic conditions, e.g., by promoting thesecretion of cytokines by antigen presenting cells that havephagocytosed the yeast (e.g., TH1-type cytokines including, but notlimited to, IFN-γ, interleukin-12 (IL-12), and IL-2, as well asproinflammatory cytokines such as IL-6). In addition, greateraccessibility to the antigens located in the cell wall is afforded bysuch culture methods. In another aspect, the use of neutral pH for someantigens allows for release of the di-sulfide bonded antigen bytreatment with dithiothreitol (DTT) that is not possible when such anantigen-expressing yeast is cultured in media at lower pH (e.g., pH 5).

In one embodiment, control of the amount of yeast glycosylation is usedto control the expression of antigens by the yeast, particularly on thesurface. The amount of yeast glycosylation can affect the immunogenicityand antigenicity of the antigen expressed on the surface, since sugarmoieties tend to be bulky. As such, the existence of sugar moieties onthe surface of yeast and its impact on the three-dimensional spacearound the target antigen(s) should be considered in the modulation ofyeast according to the invention. Any method can be used to reduce theamount of glycosylation of the yeast (or increase it, if desired). Forexample, one could use a yeast mutant strain that has been selected tohave low glycosylation (e.g., mnn1, och1 and mnn9 mutants), or one couldeliminate by mutation the glycosylation acceptor sequences on the targetantigen. Alternatively, one could use a yeast with abbreviatedglycosylation patterns, e.g., Pichia. One can also treat the yeast usingmethods that reduce or alter the glycosylation.

In one embodiment of the present invention, as an alternative toexpression of an antigen or other protein recombinantly in the yeastvehicle, a yeast vehicle is loaded intracellularly with the protein orpeptide, or with carbohydrates or other molecules that serve as anantigen and/or are useful as immunomodulatory agents or biologicalresponse modifiers according to the invention. Subsequently, the yeastvehicle, which now contains the antigen and/or other proteinsintracellularly, can be administered to an individual or loaded into acarrier such as a dendritic cell. Peptides and proteins can be inserteddirectly into yeast vehicles of the present invention by techniquesknown to those skilled in the art, such as by diffusion, activetransport, liposome fusion, electroporation, phagocytosis, freeze-thawcycles and bath sonication. Yeast vehicles that can be directly loadedwith peptides, proteins, carbohydrates, or other molecules includeintact yeast, as well as spheroplasts, ghosts or cytoplasts, which canbe loaded with antigens and other agents after production.Alternatively, intact yeast can be loaded with the antigen and/or agent,and then spheroplasts, ghosts, cytoplasts, or subcellular particles canbe prepared therefrom. Any number of antigens and/or other agents can beloaded into a yeast vehicle in this embodiment, from at least 1, 2, 3, 4or any whole integer up to hundreds or thousands of antigens and/orother agents, such as would be provided by the loading of amicroorganism or portions thereof, for example.

In another embodiment of the present invention, an antigen and/or otheragent is physically attached to the yeast vehicle. Physical attachmentof the antigen and/or other agent to the yeast vehicle can beaccomplished by any method suitable in the art, including covalent andnon-covalent association methods which include, but are not limited to,chemically crosslinking the antigen and/or other agent to the outersurface of the yeast vehicle or biologically linking the antigen and/orother agent to the outer surface of the yeast vehicle, such as by usingan antibody or other binding partner. Chemical cross-linking can beachieved, for example, by methods including glutaraldehyde linkage,photoaffinity labeling, treatment with carbodiimides, treatment withchemicals capable of linking di-sulfide bonds, and treatment with othercross-linking chemicals standard in the art. Alternatively, a chemicalcan be contacted with the yeast vehicle that alters the charge of thelipid bilayer of yeast membrane or the composition of the cell wall sothat the outer surface of the yeast is more likely to fuse or bind toantigens and/or other agent having particular charge characteristics.Targeting agents such as antibodies, binding peptides, solublereceptors, and other ligands may also be incorporated into an antigen asa fusion protein or otherwise associated with an antigen for binding ofthe antigen to the yeast vehicle.

When the antigen or other protein is expressed on or physically attachedto the surface of the yeast, spacer arms may, in one aspect, becarefully selected to optimize antigen or other protein expression orcontent on the surface. The size of the spacer arm(s) can affect howmuch of the antigen or other protein is exposed for binding on thesurface of the yeast. Thus, depending on which antigen(s) or otherprotein(s) are being used, one of skill in the art will select a spacerarm that effectuates appropriate spacing for the antigen or otherprotein on the yeast surface. In one embodiment, the spacer arm is ayeast protein of at least 450 amino acids. Spacer arms have beendiscussed in detail above.

In yet another embodiment, the yeast vehicle and the antigen or otherprotein are associated with each other by a more passive, non-specificor non-covalent binding mechanism, such as by gently mixing the yeastvehicle and the antigen or other protein together in a buffer or othersuitable formulation (e.g., admixture).

In one embodiment of the invention, the yeast vehicle and the antigen orother protein are both loaded intracellularly into a carrier such as adendritic cell or macrophage to form the therapeutic composition orvaccine of the present invention. Alternatively, an antigen or otherprotein can be loaded into a dendritic cell in the absence of the yeastvehicle.

In one embodiment, intact yeast (with or without expression ofheterologous antigens or other proteins) can be ground up or processedin a manner to produce yeast cell wall preparations, yeast membraneparticles or yeast fragments (i.e., not intact) and the yeast fragmentscan, in some embodiments, be provided with or administered with othercompositions that include antigens (e.g., DNA vaccines, protein subunitvaccines, killed or inactivated pathogens) to enhance immune responses.For example, enzymatic treatment, chemical treatment or physical force(e.g., mechanical shearing or sonication) can be used to break up theyeast into parts that are used as an adjuvant.

In one embodiment of the invention, yeast vehicles useful in theinvention include yeast vehicles that have been killed or inactivated.Killing or inactivating of yeast can be accomplished by any of a varietyof suitable methods known in the art. For example, heat inactivation ofyeast is a standard way of inactivating yeast, and one of skill in theart can monitor the structural changes of the target antigen, ifdesired, by standard methods known in the art. Alternatively, othermethods of inactivating the yeast can be used, such as chemical,electrical, radioactive or UV methods. See, for example, the methodologydisclosed in standard yeast culturing textbooks such as Methods ofEnzymology, Vol. 194, Cold Spring Harbor Publishing (1990). Any of theinactivation strategies used should take the secondary, tertiary orquaternary structure of the target antigen into consideration andpreserve such structure as to optimize its immunogenicity.

Yeast vehicles can be formulated into yeast-based immunotherapycompositions or products of the present invention, includingpreparations to be administered to a subject directly or first loadedinto a carrier such as a dendritic cell, using a number of techniquesknown to those skilled in the art. For example, yeast vehicles can bedried by lyophilization. Formulations comprising yeast vehicles can alsobe prepared by packing yeast in a cake or a tablet, such as is done foryeast used in baking or brewing operations. In addition, yeast vehiclescan be mixed with a pharmaceutically acceptable excipient, such as anisotonic buffer that is tolerated by a host or host cell. Examples ofsuch excipients include water, saline, Ringer's solution, dextrosesolution, Hank's solution, and other aqueous physiologically balancedsalt solutions. Nonaqueous vehicles, such as fixed oils, sesame oil,ethyl oleate, or triglycerides may also be used. Other usefulformulations include suspensions containing viscosity-enhancing agents,such as sodium carboxymethylcellulose, sorbitol, glycerol or dextran.Excipients can also contain minor amounts of additives, such assubstances that enhance isotonicity and chemical stability. Examples ofbuffers include phosphate buffer, bicarbonate buffer and Tris buffer,while examples of preservatives include thimerosal, m- or o-cresol,formalin and benzyl alcohol. Standard formulations can either be liquidinjectables or solids which can be taken up in a suitable liquid as asuspension or solution for injection. Thus, in a non-liquid formulation,the excipient can comprise, for example, dextrose, human serum albumin,and/or preservatives to which sterile water or saline can be added priorto administration.

In one embodiment of the present invention, a composition can includeadditional agents, which may also be referred to as biological responsemodifier compounds, or the ability to produce such agents/modifiers. Forexample, a yeast vehicle can be transfected with or loaded with at leastone antigen and at least one agent/biological response modifiercompound, or a composition of the invention can be administered inconjunction with at least one agent/biological response modifier.Biological response modifiers include adjuvants and other compounds thatcan modulate immune responses, which may be referred to asimmunomodulatory compounds, as well as compounds that modify thebiological activity of another compound or agent, such as a yeast-basedimmunotherapeutic, such biological activity not being limited to immunesystem effects. Certain immunomodulatory compounds can stimulate aprotective immune response whereas others can suppress a harmful immuneresponse, and whether an immunomodulatory is useful in combination witha given yeast-based immunotherapeutic may depend, at least in part, onthe disease state or condition to be treated or prevented, and/or on theindividual who is to be treated. Certain biological response modifierspreferentially enhance a cell-mediated immune response whereas otherspreferentially enhance a humoral immune response (i.e., can stimulate animmune response in which there is an increased level of cell-mediatedcompared to humoral immunity, or vice versa). Certain biologicalresponse modifiers have one or more properties in common with thebiological properties of yeast-based immunotherapeutics or enhance orcomplement the biological properties of yeast-based immunotherapeutics.There are a number of techniques known to those skilled in the art tomeasure stimulation or suppression of immune responses, as well as todifferentiate cell-mediated immune responses from humoral immuneresponses, and to differentiate one type of cell-mediated response fromanother (e.g., a TH17 response versus a TH1 response).

Agents/biological response modifiers useful in the invention mayinclude, but are not limited to, cytokines, chemokines, hormones,lipidic derivatives, peptides, proteins, polysaccharides, small moleculedrugs, antibodies and antigen binding fragments thereof (including, butnot limited to, anti-cytokine antibodies, anti-cytokine receptorantibodies, anti-chemokine antibodies), vitamins, polynucleotides,nucleic acid binding moieties, aptamers, and growth modulators. Somesuitable agents include, but are not limited to, IL-1 or agonists ofIL-1 or of IL-1R, anti-IL-1 or other IL-1 antagonists; IL-6 or agonistsof IL-6 or of IL-6R, anti-IL-6 or other IL-6 antagonists; IL-12 oragonists of IL-12 or of IL-12R, anti-IL-12 or other IL-12 antagonists;IL-17 or agonists of IL-17 or of IL-17R, anti-IL-17 or other IL-17antagonists; IL-21 or agonists of IL-21 or of IL-21R, anti-IL-21 orother IL-21 antagonists; IL-22 or agonists of IL-22 or of IL-22R,anti-IL-22 or other IL-22 antagonists; IL-23 or agonists of IL-23 or ofIL-23R, anti-IL-23 or other IL-23 antagonists; IL-25 or agonists ofIL-25 or of IL-25R, anti-IL-25 or other IL-25 antagonists; IL-27 oragonists of IL-27 or of IL-27R, anti-IL-27 or other IL-27 antagonists;type I interferon (including IFN-α) or agonists or antagonists of type Iinterferon or a receptor thereof; type II interferon (including IFN-γ)or agonists or antagonists of type II interferon or a receptor thereof;anti-CD40 antibody, CD40L, anti-CTLA-4 antibody (e.g., to releaseanergic T cells); T cell co-stimulators (e.g., anti-CD137, anti-CD28,anti-CD40); alemtuzumab (e.g., CamPath®), denileukin diftitox (e.g.,ONTAK®); anti-CD4; anti-CD25; anti-PD-1, anti-PD-L1, anti-PD-L2; agentsthat block FOXP3 (e.g., to abrogate the activity/kill CD4⁺/CD25⁺ Tregulatory cells); Flt3 ligand, imiquimod (Aldara™),granulocyte-macrophage colony stimulating factor (GM-CSF);granulocyte-colony stimulating factor (G-CSF), sargramostim (Leukine®);hormones including without limitation prolactin and growth hormone;Toll-like receptor (TLR) agonists, including but not limited to TLR-2agonists, TLR-4 agonists, TLR-7 agonists, and TLR-9 agonists; TLRantagonists, including but not limited to TLR-2 antagonists, TLR-4antagonists, TLR-7 antagonists, and TLR-9 antagonists; anti-inflammatoryagents and immunomodulators, including but not limited to, COX-2inhibitors (e.g., Celecoxib, NSAIDS), glucocorticoids, statins, andthalidomide and analogues thereof including IMiD™s (which are structuraland functional analogues of thalidomide (e.g., REVLIMID® (lenalidomide),ACTIMID® (pomalidomide)); proinflammatory agents, such as fungal orbacterial components or any proinflammatory cytokine or chemokine;immunotherapeutic vaccines including, but not limited to, virus-basedvaccines, bacteria-based vaccines, or antibody-based vaccines; and anyother immunomodulators, immunopotentiators, anti-inflammatory agents,and/or pro-inflammatory agents. Any combination of such agents iscontemplated by the invention, and any of such agents combined with oradministered in a protocol with (e.g., concurrently, sequentially, or inother formats with) a yeast-based immunotherapeutic is a compositionencompassed by the invention. Such agents are well known in the art.These agents may be used alone or in combination with other agentsdescribed herein.

Agents can include agonists and antagonists of a given protein orpeptide or domain thereof. As used herein, an “agonist” is any compoundor agent, including without limitation small molecules, proteins,peptides, antibodies, nucleic acid binding agents, etc., that binds to areceptor or ligand and produces or triggers a response, which mayinclude agents that mimic the action of a naturally occurring substancethat binds to the receptor or ligand. An “antagonist” is any compound oragent, including without limitation small molecules, proteins, peptides,antibodies, nucleic acid binding agents, etc., that blocks or inhibitsor reduces the action of an agonist.

Compositions of the invention can further include or can be administeredwith (concurrently, sequentially, or intermittently with) any othercompounds or compositions that are useful for preventing or treating HBVinfection or any compounds that treat or ameliorate any symptom of HBVinfection. A variety of agents are known to be useful for preventingand/or treating or ameliorating HBV infection. Such agents include, butare not limited to, anti-viral compounds, including, but not limited to,nucleotide analogue reverse transcriptase inhibitor (nRTIs). In oneaspect of the invention, suitable anti-viral compounds include, but arenot limited to: tenofovir (VIREAD®), lamivudine (EPIVIR®), adefovir(HEPSERA®), telbivudine (TYZEKA®), entecavir (BARACLUDE®), andcombinations thereof, and/or interferons, such as interferon-α2a orpegylated interferon-α2a (PEGASYS®) or interferon-λ. These agents aretypically administered for long periods of time (e.g., daily or weeklyfor up to one to five years or longer). In addition, compositions of theinvention can be used together with other immunotherapeuticcompositions, including prophylactic and/or therapeutic immunotherapy.For example, prophylactic vaccines for HBV have been commerciallyavailable since the early 1980's. These commercial vaccines arenon-infectious, subunit viral vaccines providing purified recombinanthepatitis B virus surface antigen (HBsAg), and can be administeredbeginning at birth. While no therapeutic immunotherapeutic compositionshave been approved in the U.S. for the treatment of HBV, suchcompositions can include HBV protein or epitope subunit vaccines, HBVviral vector vaccines, cytokines, and/or other immunomodulatory agents(e.g., TLR agonists, immunomodulatory drugs).

The invention also includes a kit comprising any of the compositionsdescribed herein, or any of the individual components of thecompositions described herein.

Methods for Administration or Use of Compositions of the Invention

Compositions of the Invention, which can Include any One or More (e.g.,combinations of two, three, four, five, or more) yeast-basedimmunotherapeutic compositions described herein, HBV antigens includingHBV proteins and fusion proteins, and/or recombinant nucleic acidmolecules encoding such HBV proteins or fusion proteins described above,and other compositions comprising such yeast-based compositions,antigens, proteins, fusion proteins, or recombinant molecules describedherein, can be used in a variety of in vivo and in vitro methods,including, but not limited to, to treat and/or prevent HBV infection andits sequelae, in diagnostic assays for HBV, or to produce antibodiesagainst HBV.

One embodiment of the invention relates to a method to treat chronichepatitis B virus (HBV) infection, and/or to prevent, ameliorate ortreat at least one symptom of chronic HBV infection, in an individual orpopulation of individuals. The method includes the step of administeringto an individual or a population of individuals who are chronicallyinfected with HBV one or more immunotherapeutic compositions of theinvention. In one aspect, the composition is an immunotherapeuticcomposition comprising one or more HBV antigens as described herein,which can include a yeast-based immunotherapeutic composition. In oneaspect, the composition includes a protein or fusion protein comprisingHBV antigens as described herein, and/or recombinant nucleic acidmolecule encoding such protein or fusion protein. In one embodiment, theindividual or population of individuals has chronic HBV infection. Inone aspect, the individual or population of individuals is additionallytreated with at least one other therapeutic compound useful for thetreatment of HBV infection. Such therapeutic compounds include, but arenot limited to, direct-acting antiviral drugs (e.g., those describedabove or elsewhere herein) and/or interferons and/or otherimmunotherapeutic or immunomodulatory agents. In one aspect, suchtherapeutic compounds include host-targeted therapeutics (e.g.,cyclophilin inhibitors which can interfere with viral replication, orre-entry inhibitors that can interfere with the viral life cycle(re-infection)).

“Standard Of Care” or “SOC” generally refers to the current approvedstandard of care for the treatment of a specific disease. In chronic HBVinfection, SOC may be one of several different approved therapeuticprotocols, and include, but may not be limited to, interferon therapyand/or anti-viral therapy. Currently approved anti-viral drugs for thetreatment of HBV infection include tenofovir (VIREAD®), lamivudine(EPIVIR®), adefovir (HEPSERA®), telbivudine (TYZEKA®) and entecavir(BARACLUDE®). The anti-viral drugs prescribed most often for chronic HBVinfection currently are tenofovir and entecavir. Interferon useful forthe treatment of chronic HBV infection includes a type I interferon suchas interferon-α, including, but not limited to interferon-α2 orpegylated interferon-α2 (e.g., PEGASYS®). In one embodiment, theinterferon is a type III interferon, including without limitation,interferon-λ1, interferon-λ2, and/or interferon-λ3. Theimmunotherapeutic composition of the invention can be administered priorto, concurrently with, intermittently with, and/or after one or moreanti-viral(s) and/or interferon and/or other immunotherapeutic orimmunomodulatory agents. The other therapeutic compounds may also beadministered prior to or after treatment with the immunotherapeuticcompositions of the invention.

HBV infection is typically diagnosed in an individual by detection ofHBsAg (hepatitis B virus surface antigen) and/or HBeAg (e-antigen) inthe blood of the infected individual. The detection of HBeAg in theserum reflects active viral replication, and clinical outcome ofinfection can be correlated with e-antigen status, although long-termremission (or cure) is better predicted using HBsAg seroconversion whenusing current therapies (see below). Detection of IgM core antibody mayalso be used to detect acute HBV infection during the first 6-12 monthsof infection. Persistence of HBsAg in the blood for more than 6 monthstypically identifies chronic HBV infection. In addition, chronic HBVinfection can be diagnosed by identifying HBV DNA (>2000 IU/ml), whichcan be combined with detection or identification of elevated serumalanine aminotransferase (ALT) and/or aspartate aminotrasferase (AST)levels (e.g., more than twice the upper limit of normal).

Recovery from the viral infection (complete response, or the endpointfor a treatment of HBV) is determined by HBeAg/HBsAg seroconversion,which is loss of HBeAg and HBsAg, respectively, and the development ofantibodies against the hepatitis B surface antigen (anti-HBs) and/orantibodies against HBeAg. Clinical studies have defined seroconversion,or a protective antibody (anti-HBs) level as: (a) 10 or more sampleratio units (SRU) as determined by radioimmunoassay; (b) a positiveresult as determined by enzyme immunoassay; or (c) detection of anantibody concentration of ≦10 mIU/ml (10 SRU is comparable to 10 mIU/mLof antibody). Seroconversion can take years to develop in a chronicallyinfected patient under current standard of care treatment (i.e.,anti-viral drugs or interferon). Patients can also be monitored for lossor marked reduction of viral DNA (below detectable levels by PCR or<2000 IU/ml), normalization of serum alanine aminotransferase (ALT)levels, and improvement in liver inflammation and fibrosis. “ALT” is awell-validated measure of hepatic injury and serves as a surrogate forhepatic inflammation. In prior large hepatitis trials, reductions and/ornormalization of ALT levels (ALT normalization) have been shown tocorrelate with improved liver function and reduced liver fibrosis asdetermined by serial biopsy.

Another embodiment of the invention relates to a method to immunize anindividual or population of individuals against HBV in order to preventHBV infection, prevent chronic HBV infection, and/or reduce the severityof HBV infection in the individual or population of individuals. Themethod includes the step of administering to an individual or populationof individuals that is not infected with HBV (or believed not to beinfected with HBV), a composition of the invention. In one aspect, thecomposition is an immunotherapeutic composition comprising one or moreHBV antigens as described herein, including one or more yeast-basedimmunotherapeutic compositions. In one aspect, the composition includesa fusion protein comprising HBV antigens as described herein, orrecombinant nucleic acid molecule encoding such fusion protein.

As used herein, the phrase “treat” HBV infection, or any permutationthereof (e.g., “treated for HBV infection”, etc.) generally refers toapplying or administering a composition of the invention once theinfection (acute or chronic) has occurred, with the goal of reduction orelimination of detectable viral titer (e.g., reduction of viral DNA(below detectable levels by PCR or <2000 IU/ml)), reachingseroconversion (development of antibodies against HBsAg and/or HBeAg andconcurrent loss or reduction of these proteins from the serum),reduction in at least one symptom resulting from the infection in theindividual, delaying or preventing the onset and/or severity of symptomsand/or downstream sequelae caused by the infection, reduction of organor physiological system damage (e.g., cirrhosis) resulting from theinfection (e.g., reduction of abnormal ALT levels, reduction of liverinflammation, reduction of liver fibrosis), prevention and/or reductionin the frequency and incidence of hepatocellular carcinoma (HCC),improvement in organ or system function that was negatively impacted bythe infection (normalization of serum ALT levels, improvement in liverinflammation, improvement in liver fibrosis), improvement of immuneresponses against the infection, improvement of long term memory immuneresponses against the infection, reduced reactivation of HBV virus,and/or improved general health of the individual or population ofindividuals.

In one aspect, a goal of treatment is sustained viral clearance for atleast 6 months after the completion of therapy. In one aspect, a goal oftreatment is the loss of detectable serum HBeAg and/or HBsAg proteins.In one aspect, a goal of treatment is the development of antibodiesagainst the hepatitis B surface antigen (anti-HBs) and/or antibodiesagainst HBeAg. In one aspect, the goal of treatment is seroconversion,which may be defined by: (a) 10 or more sample ratio units (SRU) asdetermined by radioimmunoassay; (b) a positive result as determined byenzyme immunoassay; or (c) detection of an antibody concentration of ≦10mIU/ml (10 SRU is comparable to 10 mIU/mL of antibody).

To “prevent” HBV infection, or any permutation thereof (e.g.,“prevention of HBV infection”, etc.), generally refers to applying oradministering a composition of the invention before an infection withHBV has occurred, with the goal of preventing infection by HBV,preventing chronic infection by HBV (i.e., enabling an individual toclear an acute HBV infection without further intervention), or, shouldthe infection later occur, at least reducing the severity, and/or lengthof infection and/or the physiological damage caused by the chronicinfection, including preventing or reducing the severity or incidence ofat least one symptom resulting from the infection in the individual,and/or delaying or preventing the onset and/or severity of symptomsand/or downstream sequelae caused by the infection, in an individual orpopulation of individuals. In one aspect, the present invention can beused to prevent chronic HBV infection, such as by enabling an individualwho becomes acutely infected with HBV subsequent to administration of acomposition of the invention to clear the infection and not becomechronically infected.

The present invention includes the delivery (administration,immunization) of one or more immunotherapeutic compositions of theinvention, including a yeast-based immunotherapy composition, to asubject. The administration process can be performed ex vivo or in vivo,but is typically performed in vivo. Ex vivo administration refers toperforming part of the regulatory step outside of the patient, such asadministering a composition of the present invention to a population ofcells (dendritic cells) removed from a patient under conditions suchthat a yeast vehicle, antigen(s) and any other agents or compositionsare loaded into the cell, and returning the cells to the patient. Thetherapeutic composition of the present invention can be returned to apatient, or administered to a patient, by any suitable mode ofadministration.

Administration of a composition can be systemic, mucosal and/or proximalto the location of the target site (e.g., near a site of infection).Suitable routes of administration will be apparent to those of skill inthe art, depending on the type of condition to be prevented or treated,the antigen used, and/or the target cell population or tissue. Variousacceptable methods of administration include, but are not limited to,intravenous administration, intraperitoneal administration,intramuscular administration, intranodal administration, intracoronaryadministration, intraarterial administration (e.g., into a carotidartery), subcutaneous administration, transdermal delivery,intratracheal administration, intraarticular administration,intraventricular administration, inhalation (e.g., aerosol),intracranial, intraspinal, intraocular, aural, intranasal, oral,pulmonary administration, impregnation of a catheter, and directinjection into a tissue. In one aspect, routes of administrationinclude: intravenous, intraperitoneal, subcutaneous, intradermal,intranodal, intramuscular, transdermal, inhaled, intranasal, oral,intraocular, intraarticular, intracranial, and intraspinal. Parenteraldelivery can include intradermal, intramuscular, intraperitoneal,intrapleural, intrapulmonary, intravenous, subcutaneous, atrial catheterand venal catheter routes. Aural delivery can include ear drops,intranasal delivery can include nose drops or intranasal injection, andintraocular delivery can include eye drops. Aerosol (inhalation)delivery can also be performed using methods standard in the art (see,for example, Stribling et al., Proc. Natl. Acad. Sci. USA189:11277-11281, 1992). Other routes of administration that modulatemucosal immunity may be useful in the treatment of viral infections.Such routes include bronchial, intradermal, intramuscular, intranasal,other inhalatory, rectal, subcutaneous, topical, transdermal, vaginaland urethral routes. In one aspect, an immunotherapeutic composition ofthe invention is administered subcutaneously.

With respect to the yeast-based immunotherapy compositions of theinvention, in general, a suitable single dose is a dose that is capableof effectively providing a yeast vehicle and an antigen (if included) toa given cell type, tissue, or region of the patient body in an amounteffective to elicit an antigen-specific immune response against one ormore HBV antigens or epitopes, when administered one or more times overa suitable time period. For example, in one embodiment, a single dose ofa yeast vehicle of the present invention is from about 1×10⁵ to about5×10⁷ yeast cell equivalents per kilogram body weight of the organismbeing administered the composition. In one aspect, a single dose of ayeast vehicle of the present invention is from about 0.1 Y.U. (1×10⁶cells) to about 100 Y.U. (1×10⁹ cells) per dose (i.e., per organism),including any interim dose, in increments of 0.1×10⁶ cells (i.e.,1.1×10⁶, 1.2×10⁶, 1.3×10⁶ . . . ). In one embodiment, doses includedoses between 1 Y.U and 40 Y.U., doses between 1 Y.U. and 50 Y.U., dosesbetween 1 Y.U. and 60 Y.U., doses between 1 Y.U. and 70 Y.U., or dosesbetween 1 Y.U. and 80 Y.U., and in one aspect, between 10 Y.U. and 40Y.U., 50 Y.U., 60 Y.U., 70 Y.U., or 80 Y.U. In one embodiment, the dosesare administered at different sites on the individual but during thesame dosing period. For example, a 40 Y.U. dose may be administered viaby injecting 10 Y.U. doses to four different sites on the individualduring one dosing period, or a 20 Y.U. dose may be administered byinjecting 5 Y.U. doses to four different sites on the individual, or byinjecting 10 Y.U. doses to two different sites on the individual, duringthe same dosing period. The invention includes administration of anamount of the yeast-based immunotherapy composition (e.g., 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 Y.U. or more)at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different sites on anindividual to form a single dose.

“Boosters” or “boosts” of a therapeutic composition are administered,for example, when the immune response against the antigen has waned oras needed to provide an immune response or induce a memory responseagainst a particular antigen or antigen(s). Boosters can be administeredfrom about 1, 2, 3, 4, 5, 6, 7, or 8 weeks apart, to monthly, tobimonthly, to quarterly, to annually, to several years after theoriginal administration. In one embodiment, an administration scheduleis one in which from about 1×10⁵ to about 5×10⁷ yeast cell equivalentsof a composition per kg body weight of the organism is administered atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times over a time period offrom weeks, to months, to years. In one embodiment, the doses areadministered weekly for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more doses,followed by monthly doses as needed to achieve the desired inhibition orelimination of the HBV virus. For example, the doses can be administereduntil the individual achieves seroconversion, until HBV DNA titers fallbelow 2000 IU/ml, and/or until ALT levels normalize. In one embodiment,the doses are administered in a 4-weekly protocol (every 4 weeks, or onday 1, week 4, week 8, week 12, etc., for between 2 and 10 doses orlonger as determined by the clinician). Additional doses can beadministered even after the individual achieves seroconversion, ifdesired, although such dosing may not be necessary.

With respect to administration of yeast-based immunotherapeuticcompositions described herein, a single composition can be administeredto an individual or population of individuals or combination of suchcompositions can be administered. For example, the invention providesseveral “single protein” compositions or compositions directed against aparticular genotype, as well as multi-protein compositions andcompositions that target multiple genotypes, or sub-genotypes.Accordingly, two or more compositions can be selected in a “spice rack”approach to most effectively prevent or treat HBV infection in a givenindividual or population of individuals.

In one aspect of the invention, one or more additional therapeuticagents are administered sequentially with the yeast-based immunotherapycomposition. In another embodiment, one or more additional therapeuticagents are administered before the yeast-based immunotherapy compositionis administered. In another embodiment, one or more additionaltherapeutic agents are administered after the yeast-based immunotherapycomposition is administered. In one embodiment, one or more additionaltherapeutic agents are administered in alternating doses with theyeast-based immunotherapy composition, or in a protocol in which theyeast-based composition is administered at prescribed intervals inbetween or with one or more consecutive doses of the additional agents,or vice versa. In one embodiment, the yeast-based immunotherapycomposition is administered in one or more doses over a period of timeprior to commencing the administration of the additional agents. Inother words, the yeast-based immunotherapeutic composition isadministered as a monotherapy for a period of time, and then the agentadministration is added, either concurrently with new doses ofyeast-based immunotherapy, or in an alternating fashion with yeast-basedimmunotherapy. Alternatively, the agent may be administered for a periodof time prior to beginning administration of the yeast-basedimmunotherapy composition. In one aspect, the yeast is engineered toexpress or carry the agent, or a different yeast is engineered orproduced to express or carry the agent.

In one aspect of the invention, when a treatment course of interferon oranti-viral compound therapy begins, additional doses of theimmunotherapeutic composition are administered over the same period oftime, or for at least a portion of that time, and may continue to beadministered once the course of interferon or anti-viral compound hasended. However, the dosing schedule for the immunotherapy over theentire period may be, and is expected to typically be, different thanthat for the interferon or the anti-viral compound. For example, theimmunotherapeutic composition may be administered on the same days or atleast 3-4 days after the last given (most recent) dose of interferon oranti-viral (or any suitable number of days after the last dose), and maybe administered daily, weekly, biweekly, monthly, bimonthly, or every3-6 months, or at longer intervals as determined by the physician.During an initial period of monotherapy administration of theimmunotherapeutic composition, if utilized, the immunotherapeuticcomposition is preferably administered weekly for between 4 and 12weeks, followed by monthly administration (regardless of when theadditional interferon or anti-viral therapy is added into the protocol).In one aspect, the immunotherapeutic composition is administered weeklyfor four or five weeks, followed by monthly administration thereafter,until conclusion of the complete treatment protocol.

In aspects of the invention, an immunotherapeutic composition and otheragents can be administered together (concurrently). As used herein,concurrent use does not necessarily mean that all doses of all compoundsare administered on the same day at the same time. Rather, concurrentuse means that each of the therapy components (e.g., immunotherapy andinterferon therapy, or immunotherapy and anti-viral therapy) are startedat approximately the same period (within hours, or up to 1-7 days ofeach other) and are administered over the same general period of time,noting that each component may have a different dosing schedule (e.g.,interferon weekly, immunotherapy monthly, anti-viral daily or weekly).In addition, before or after the concurrent administration period, anyone of the agents or immunotherapeutic compositions can be administeredwithout the other agent(s).

It is contemplated by the present invention that the use of animmunotherapeutic composition of the invention with an anti-viral suchas tenofovir or entecavir will enable a shorter time course for the useof the anti-viral drug. Similar results are expected when combining animmunotherapeutic of the invention with interferon. Dosing requirementsfor the anti-viral or interferon may also be reduced or modified as aresult of combination with the immunotherapeutic of the invention togenerally improve the tolerance of the patient for the drug. Inaddition, it is contemplated that the immunotherapeutic composition ofthe invention will enable seroconversion or sustained viral responsesfor patients in whom anti-viral therapy alone fails to achieve theseendpoints. In other words, more patients will achieve seroconversionwhen an immunotherapeutic composition of the invention is combined withan anti-viral or interferon than will achieve seroconversion by usinganti-virals or interferon alone. Under current SOC for HBV infection,anti-virals may be administered for 6 months to one year, two years,three years, four years, five years, or longer (e.g., indefinitely). Bycombining such therapy with an immunotherapeutic composition of theinvention, the time for the administration of the anti-viral may bereduced by several months or years. It is contemplated that use of theimmunotherapeutic compositions of the present invention, as amonotherapy or in combination with anti-viral and/or immunomodulatoryapproaches will be effective to achieve loss of HBsAg and/or HBeAg;HBeAg seroconversion, HBsAg seroconversion, or complete seroconversion;and in many individuals, sustained viral clearance for at least 6 monthsafter the completion of therapy. In some patients, immunotherapyaccording to the present invention, when used as a monotherapy or incombination with anti-viral and/or immunomodulatory approaches, mayachieve loss of HBsAg and/or HBeAg, but not achieve seroconversion(development of anti-HBs or anti-HBeAg). In this scenario, it is anembodiment of the invention to additionally use, alone or in combinationwith the yeast-based immunotherapy of the invention and/or anti-viralsor other immunomodulatory agents, an agent such as the currentprophylactic recombinant HBV subunit vaccine, in order to achievecomplete response in the patient.

As used herein, the term “anti-viral” refers to any compound or drug,typically a small-molecule inhibitor or antibody, which targets one ormore steps in the virus life cycle with direct anti-viral therapeuticeffects. In one embodiment of the invention, the anti-viral compound ordrug to be administered in the same therapeutic protocol with animmunotherapeutic composition of the invention is selected fromtenofovir (VIREAD®), lamivudine (EPIVIR®), adefovir (HEPSERA®),telbivudine (TYZEKA®) and entecavir (BARACLUDE®), or any analog orderivative thereof, or any composition comprising or containing suchcompound, drug, analog or derivative.

Tenofovir (tenofovir disoproxil fumarate or TDF), or({[(2R)-1-(6-amino-9H-purin-9-yl)propan-2-yl]oxy}methyl)phosphonic acid,is a nucleotide analogue reverse transcriptase inhibitor (nRTIs). Forthe treatment of HBV infection, tenofovir is typically administered toadults as a pill taken at a dose of 300 mg (tenofovir disproxilfumarate) once daily. Dosage for pediatric patients is based on bodyweight of the patient (8 mg per kg body weight, up to 300 mg once daily)and may be provided as tablet or oral powder.

Lamivudine, or 2′,3′-dideoxy-3′-thiacytidine, commonly called 3TC, is apotent nucleoside analog reverse transcriptase inhibitor (nRTI). For thetreatment of HBV infection, lamivudine is administered as a pill or oralsolution taken at a dose of 100 mg once a day (1.4-2 mg/lb. twice a dayfor children 3 months to 12 years old).

Adefovir (adefovir dipivoxil), or9-[2-[[bis[(pivaloyloxy)methoxy]-phosphinyl]-methoxy]ethyl]adenine, isan orally-administered nucleotide analog reverse transcriptase inhibitor(ntRTI). For the treatment of HBV infection, adefovir is administered asa pill taken at a dose of 10 mg once daily.

Telbivudine, or1-(2-deoxy-(β-L-erythro-pentofuranosyl)-5-methylpyrimidine-2,4(1H,3H)-dione,is a synthetic thymidine nucleoside analogue (the L-isomer ofthymidine). For the treatment of HBV infection, telbivudine isadministered as a pill or oral solution taken at a dose of 600 mg oncedaily.

Entecavir, or2-Amino-9-[(1S,3R,4S)-4-hydroxy-3-(hydroxymethyl)-2-methylidenecyclopentyl]-6,9-dihydro-3H-purin-6-one,is a nucleoside analog (guanine analogue) that inhibits reversetranscription, DNA replication and transcription of the virus. For thetreatment of HBV infection, entecavir is administered as a pill or oralsolution taken at a dose of 0.5 mg once daily (1 mg daily forlamivudine-refractory or telbivudine resistance mutations).

In one embodiment of the invention, the interferon to be administered ina therapeutic protocol with an immunotherapeutic composition of theinvention is an interferon, and in one aspect, interferon-α, and in oneaspect, interferon-α2b (administered by subcutaneous injection 3 timesper week); or pegylated interferon-α2a (e.g. PEGASYS®). As used herein,the term “interferon” refers to a cytokine that is typically produced bycells of the immune system and by a wide variety of cells in response tothe presence of double-stranded RNA. Interferons assist the immuneresponse by inhibiting viral replication within host cells, activatingnatural killer cells and macrophages, increasing antigen presentation tolymphocytes, and inducing the resistance of host cells to viralinfection. Type I interferons include interferon-α. Type III interferonsinclude interferon-2. Interferons useful in the methods of the presentinvention include any type I or type III interferon, includinginterferon-α, interferon-α2, and in one aspect, longer lasting forms ofinterferon, including, but not limited to, pegylated interferons,interferon fusion proteins (interferon fused to albumin), andcontrolled-release formulations comprising interferon (e.g., interferonin microspheres or interferon with polyaminoacid nanoparticles). Oneinterferon, PEGASYS®, pegylated interferon-α2a, is a covalent conjugateof recombinant interferon-α2a (approximate molecular weight [MW] 20,000daltons) with a single branched bis-monomethoxy polyethylene glycol(PEG) chain (approximate MW 40,000 daltons). The PEG moiety is linked ata single site to the interferon-α moiety via a stable amide bond tolysine. Pegylated interferon-α2a has an approximate molecular weight of60,000 daltons.

Interferon is typically administered by intramuscular or subcutaneousinjection, and can be administered in a dose of between 3 and 10 millionunits, with 3 million units being preferred in one embodiment. Doses ofinterferon are administered on a regular schedule, which can vary from1, 2, 3, 4, 5, or 6 times a week, to weekly, biweekly, every threeweeks, or monthly. A typical dose of interferon that is currentlyavailable is provided weekly, and that is a preferred dosing schedulefor interferon, according to the present invention. For the treatment ofHBV, pegylated interferon-α2a is currently administered subcutaneouslyonce a week at a dose of 180 mg (1.0 ml viral or 0.5 ml prefilledsyringe), for a total of 48 weeks. The dose amount and timing can bevaried according to the preferences and recommendations of thephysician, as well as according to the recommendations for theparticular interferon being used, and it is within the abilities ofthose of skill in the art to determine the proper dose. It iscontemplated that by using interferon therapy together with animmunotherapeutic composition of the invention, the dose strength and/ornumber of doses of interferon (length of time on interferon and/orintervals between doses of interferon) can be reduced.

In the method of the present invention, compositions and therapeuticcompositions can be administered to animal, including any vertebrate,and particularly to any member of the Vertebrate class, Mammalia,including, without limitation, primates, rodents, livestock and domesticpets. Livestock include mammals to be consumed or that produce usefulproducts (e.g., sheep for wool production). Mammals to treat or protectinclude humans, dogs, cats, mice, rats, goats, sheep, cattle, horses andpigs.

An “individual” is a vertebrate, such as a mammal, including withoutlimitation a human. Mammals include, but are not limited to, farmanimals, sport animals, pets, primates, mice and rats. The term“individual” can be used interchangeably with the term “animal”,“subject” or “patient”.

General Techniques Useful in the Invention

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry,nucleic acid chemistry, and immunology, which are well known to thoseskilled in the art. Such techniques are explained fully in theliterature, such as, Methods of Enzymology, Vol. 194, Guthrie et al.,eds., Cold Spring Harbor Laboratory Press (1990); Biology and activitiesof yeasts, Skinner, et al., eds., Academic Press (1980); Methods inyeast genetics: a laboratory course manual, Rose et al., Cold SpringHarbor Laboratory Press (1990); The Yeast Saccharomyces: Cell Cycle andCell Biology, Pringle et al., eds., Cold Spring Harbor Laboratory Press(1997); The Yeast Saccharomyces: Gene Expression, Jones et al., eds.,Cold Spring Harbor Laboratory Press (1993); The Yeast Saccharomyces:Genome Dynamics, Protein Synthesis, and Energetics, Broach et al., eds.,Cold Spring Harbor Laboratory Press (1992); Molecular Cloning: ALaboratory Manual, second edition (Sambrook et al., 1989) and MolecularCloning: A Laboratory Manual, third edition (Sambrook and Russel, 2001),(jointly referred to herein as “Sambrook”); Current Protocols inMolecular Biology (F. M. Ausubel et al., eds., 1987, includingsupplements through 2001); PCR: The Polymerase Chain Reaction, (Mulliset al., eds., 1994); Harlow and Lane (1988), Antibodies, A LaboratoryManual, Cold Spring Harbor Publications, New York; Harlow and Lane(1999) Using Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (jointly referred to hereinas “Harlow and Lane”), Beaucage et al. eds., Current Protocols inNucleic Acid Chemistry, John Wiley & Sons, Inc., New York, 2000);Casarett and Doull's Toxicology The Basic Science of Poisons, C.Klaassen, ed., 6th edition (2001), and Vaccines, S. Plotkin and W.Orenstein, eds., 3rd edition (1999).

GENERAL DEFINITIONS

A “TARMOGEN™” (GlobeImmune, Inc., Louisville, Colo.) generally refers toa yeast vehicle expressing one or more heterologous antigensextracellularly (on its surface), intracellularly (internally orcytosolically) or both extracellularly and intracellularly. TARMOGEN®products have been generally described (see, e.g., U.S. Pat. No.5,830,463). Certain yeast-based immunotherapy compositions, and methodsof making and generally using the same, are also described in detail,for example, in U.S. Pat. No. 5,830,463, U.S. Pat. No. 7,083,787, U.S.Pat. No. 7,736,642, Stubbs et al., Nat. Med. 7:625-629 (2001), Lu etal., Cancer Research 64:5084-5088 (2004), and in Bernstein et al.,Vaccine 2008 Jan. 24; 26(4):509-21, each of which is incorporated hereinby reference in its entirety.

As used herein, the term “analog” refers to a chemical compound that isstructurally similar to another compound but differs slightly incomposition (as in the replacement of one atom by an atom of a differentelement or in the presence of a particular functional group, or thereplacement of one functional group by another functional group). Thus,an analog is a compound that is similar or comparable in function andappearance, but has a different structure or origin with respect to thereference compound.

The terms “substituted”, “substituted derivative” and “derivative”, whenused to describe a compound, means that at least one hydrogen bound tothe unsubstituted compound is replaced with a different atom or achemical moiety.

Although a derivative has a similar physical structure to the parentcompound, the derivative may have different chemical and/or biologicalproperties than the parent compound. Such properties can include, butare not limited to, increased or decreased activity of the parentcompound, new activity as compared to the parent compound, enhanced ordecreased bioavailability, enhanced or decreased efficacy, enhanced ordecreased stability in vitro and/or in vivo, and/or enhanced ordecreased absorption properties.

In general, the term “biologically active” indicates that a compound(including a protein or peptide) has at least one detectable activitythat has an effect on the metabolic or other processes of a cell ororganism, as measured or observed in vivo (i.e., in a naturalphysiological environment) or in vitro (i.e., under laboratoryconditions).

According to the present invention, the term “modulate” can be usedinterchangeably with “regulate” and refers generally to upregulation ordownregulation of a particular activity. As used herein, the term“upregulate” can be used generally to describe any of: elicitation,initiation, increasing, augmenting, boosting, improving, enhancing,amplifying, promoting, or providing, with respect to a particularactivity. Similarly, the term “downregulate” can be used generally todescribe any of: decreasing, reducing, inhibiting, ameliorating,diminishing, lessening, blocking, or preventing, with respect to aparticular activity.

In one embodiment of the present invention, any of the amino acidsequences described herein can be produced with from at least one, andup to about 20, additional heterologous amino acids flanking each of theC- and/or N-terminal ends of the specified amino acid sequence. Theresulting protein or polypeptide can be referred to as “consistingessentially of” the specified amino acid sequence. According to thepresent invention, the heterologous amino acids are a sequence of aminoacids that are not naturally found (i.e., not found in nature, in vivo)flanking the specified amino acid sequence, or that are not related tothe function of the specified amino acid sequence, or that would not beencoded by the nucleotides that flank the naturally occurring nucleicacid sequence encoding the specified amino acid sequence as it occurs inthe gene, if such nucleotides in the naturally occurring sequence weretranslated using standard codon usage for the organism from which thegiven amino acid sequence is derived. Similarly, the phrase “consistingessentially of”, when used with reference to a nucleic acid sequenceherein, refers to a nucleic acid sequence encoding a specified aminoacid sequence that can be flanked by from at least one, and up to asmany as about 60, additional heterologous nucleotides at each of the 5′and/or the 3′ end of the nucleic acid sequence encoding the specifiedamino acid sequence. The heterologous nucleotides are not naturallyfound (i.e., not found in nature, in vivo) flanking the nucleic acidsequence encoding the specified amino acid sequence as it occurs in thenatural gene or do not encode a protein that imparts any additionalfunction to the protein or changes the function of the protein havingthe specified amino acid sequence.

According to the present invention, the phrase “selectively binds to”refers to the ability of an antibody, antigen-binding fragment orbinding partner of the present invention to preferentially bind tospecified proteins. More specifically, the phrase “selectively binds”refers to the specific binding of one protein to another (e.g., anantibody, fragment thereof, or binding partner to an antigen), whereinthe level of binding, as measured by any standard assay (e.g., animmunoassay), is statistically significantly higher than the backgroundcontrol for the assay. For example, when performing an immunoassay,controls typically include a reaction well/tube that contain antibody orantigen binding fragment alone (i.e., in the absence of antigen),wherein an amount of reactivity (e.g., non-specific binding to the well)by the antibody or antigen-binding fragment thereof in the absence ofthe antigen is considered to be background. Binding can be measuredusing a variety of methods standard in the art including enzymeimmunoassays (e.g., ELISA, immunoblot assays, etc.).

Reference to a protein or polypeptide used in the present inventionincludes full-length proteins, fusion proteins, or any fragment, domain,conformational epitope, or homologue of such proteins, includingfunctional domains and immunological domains of proteins. Morespecifically, an isolated protein, according to the present invention,is a protein (including a polypeptide or peptide) that has been removedfrom its natural milieu (i.e., that has been subject to humanmanipulation) and can include purified proteins, partially purifiedproteins, recombinantly produced proteins, and synthetically producedproteins, for example. As such, “isolated” does not reflect the extentto which the protein has been purified. Preferably, an isolated proteinof the present invention is produced recombinantly. According to thepresent invention, the terms “modification” and “mutation” can be usedinterchangeably, particularly with regard to the modifications/mutationsto the amino acid sequence of proteins or portions thereof (or nucleicacid sequences) described herein.

As used herein, the term “homologue” is used to refer to a protein orpeptide which differs from a naturally occurring protein or peptide(i.e., the “prototype” or “wild-type” protein) by minor modifications tothe naturally occurring protein or peptide, but which maintains thebasic protein and side chain structure of the naturally occurring form.Such changes include, but are not limited to: changes in one or a fewamino acid side chains; changes one or a few amino acids, includingdeletions (e.g., a truncated version of the protein or peptide)insertions and/or substitutions; changes in stereochemistry of one or afew atoms; and/or minor derivatizations, including but not limited to:methylation, glycosylation, phosphorylation, acetylation,myristoylation, prenylation, palmitation, amidation and/or addition ofglycosylphosphatidyl inositol. A homologue can have enhanced, decreased,or substantially similar properties as compared to the naturallyoccurring protein or peptide. A homologue can include an agonist of aprotein or an antagonist of a protein. Homologues can be produced usingtechniques known in the art for the production of proteins including,but not limited to, direct modifications to the isolated, naturallyoccurring protein, direct protein synthesis, or modifications to thenucleic acid sequence encoding the protein using, for example, classicor recombinant DNA techniques to effect random or targeted mutagenesis.

A homologue of a given protein may comprise, consist essentially of, orconsist of, an amino acid sequence that is at least about 45%, or atleast about 50%, or at least about 55%, or at least about 60%, or atleast about 65%, or at least about 70%, or at least about 75%, or atleast about 80%, or at least about 85%, or at least about 90%, or atleast about 91% identical, or at least about 92% identical, or at leastabout 93% identical, or at least about 94% identical, or at least about95% identical, or at least about 96% identical, or at least about 97%identical, or at least about 98% identical, or at least about 99%identical (or any percent identity between 45% and 99%, in whole integerincrements), to the amino acid sequence of the reference protein. In oneembodiment, the homologue comprises, consists essentially of, orconsists of, an amino acid sequence that is less than 100% identical,less than about 99% identical, less than about 98% identical, less thanabout 97% identical, less than about 96% identical, less than about 95%identical, and so on, in increments of 1%, to less than about 70%identical to the naturally occurring amino acid sequence of thereference protein.

A homologue may include proteins or domains of proteins that are “nearfull-length”, which means that such a homologue differs from thefull-length protein, functional domain or immunological domain (as suchprotein, functional domain or immunological domain is described hereinor otherwise known or described in a publicly available sequence) by theaddition of or deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acidsfrom the N- and/or the C-terminus of such full-length protein orfull-length functional domain or full-length immunological domain.

As used herein, unless otherwise specified, reference to a percent (%)identity refers to an evaluation of homology which is performed using:(1) a BLAST 2.0 Basic BLAST homology search using blastp for amino acidsearches and blastn for nucleic acid searches with standard defaultparameters, wherein the query sequence is filtered for low complexityregions by default (described in Altschul, S. F., Madden, T. L.,Schààffer, A. A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D. J.(1997) “Gapped BLAST and PSI-BLAST: a new generation of protein databasesearch programs.” Nucleic Acids Res. 25:3389-3402, incorporated hereinby reference in its entirety); (2) a BLAST 2 alignment (using theparameters described below); (3) and/or PSI-BLAST with the standarddefault parameters (Position-Specific Iterated BLAST. It is noted thatdue to some differences in the standard parameters between BLAST 2.0Basic BLAST and BLAST 2, two specific sequences might be recognized ashaving significant homology using the BLAST 2 program, whereas a searchperformed in BLAST 2.0 Basic BLAST using one of the sequences as thequery sequence may not identify the second sequence in the top matches.In addition, PSI-BLAST provides an automated, easy-to-use version of a“profile” search, which is a sensitive way to look for sequencehomologues. The program first performs a gapped BLAST database search.The PSI-BLAST program uses the information from any significantalignments returned to construct a position-specific score matrix, whichreplaces the query sequence for the next round of database searching.Therefore, it is to be understood that percent identity can bedetermined by using any one of these programs.

Two specific sequences can be aligned to one another using BLAST 2sequence as described in Tatusova and Madden, (1999), “Blast 2sequences—a new tool for comparing protein and nucleotide sequences”,FEMS Microbiol Lett. 174:247-250, incorporated herein by reference inits entirety. BLAST 2 sequence alignment is performed in blastp orblastn using the BLAST 2.0 algorithm to perform a Gapped BLAST search(BLAST 2.0) between the two sequences allowing for the introduction ofgaps (deletions and insertions) in the resulting alignment. For purposesof clarity herein, a BLAST 2 sequence alignment is performed using thestandard default parameters as follows.

-   -   For blastn, using 0 BLOSUM62 matrix:    -   Reward for match=1    -   Penalty for mismatch=−2    -   Open gap (5) and extension gap (2) penalties    -   gap x_dropoff (50) expect (10) word size (11) filter (on)    -   For blastp, using 0 BLOSUM62 matrix:    -   Open gap (11) and extension gap (1) penalties    -   gap x_dropoff (50) expect (10) word size (3) filter (on).

An isolated nucleic acid molecule is a nucleic acid molecule that hasbeen removed from its natural milieu (i.e., that has been subject tohuman manipulation), its natural milieu being the genome or chromosomein which the nucleic acid molecule is found in nature. As such,“isolated” does not necessarily reflect the extent to which the nucleicacid molecule has been purified, but indicates that the molecule doesnot include an entire genome or an entire chromosome in which thenucleic acid molecule is found in nature. An isolated nucleic acidmolecule can include a gene. An isolated nucleic acid molecule thatincludes a gene is not a fragment of a chromosome that includes suchgene, but rather includes the coding region and regulatory regionsassociated with the gene, but no additional genes that are naturallyfound on the same chromosome. An isolated nucleic acid molecule can alsoinclude a specified nucleic acid sequence flanked by (i.e., at the 5′and/or the 3′ end of the sequence) additional nucleic acids that do notnormally flank the specified nucleic acid sequence in nature (i.e.,heterologous sequences). Isolated nucleic acid molecule can include DNA,RNA (e.g., mRNA), or derivatives of either DNA or RNA (e.g., cDNA).Although the phrase “nucleic acid molecule” primarily refers to thephysical nucleic acid molecule and the phrase “nucleic acid sequence”primarily refers to the sequence of nucleotides on the nucleic acidmolecule, the two phrases can be used interchangeably, especially withrespect to a nucleic acid molecule, or a nucleic acid sequence, beingcapable of encoding a protein or domain of a protein.

A recombinant nucleic acid molecule is a molecule that can include atleast one of any nucleic acid sequence encoding any one or more proteinsdescribed herein operatively linked to at least one of any transcriptioncontrol sequence capable of effectively regulating expression of thenucleic acid molecule(s) in the cell to be transfected. Although thephrase “nucleic acid molecule” primarily refers to the physical nucleicacid molecule and the phrase “nucleic acid sequence” primarily refers tothe sequence of nucleotides on the nucleic acid molecule, the twophrases can be used interchangeably, especially with respect to anucleic acid molecule, or a nucleic acid sequence, being capable ofencoding a protein. In addition, the phrase “recombinant molecule”primarily refers to a nucleic acid molecule operatively linked to atranscription control sequence, but can be used interchangeably with thephrase “nucleic acid molecule” which is administered to an animal.

A recombinant nucleic acid molecule includes a recombinant vector, whichis any nucleic acid sequence, typically a heterologous sequence, whichis operatively linked to the isolated nucleic acid molecule encoding afusion protein of the present invention, which is capable of enablingrecombinant production of the fusion protein, and which is capable ofdelivering the nucleic acid molecule into a host cell according to thepresent invention. Such a vector can contain nucleic acid sequences thatare not naturally found adjacent to the isolated nucleic acid moleculesto be inserted into the vector. The vector can be either RNA or DNA,either prokaryotic or eukaryotic, and preferably in the presentinvention, is a virus or a plasmid. Recombinant vectors can be used inthe cloning, sequencing, and/or otherwise manipulating of nucleic acidmolecules, and can be used in delivery of such molecules (e.g., as in aDNA composition or a viral vector-based composition). Recombinantvectors are preferably used in the expression of nucleic acid molecules,and can also be referred to as expression vectors. Preferred recombinantvectors are capable of being expressed in a transfected host cell.

In a recombinant molecule of the present invention, nucleic acidmolecules are operatively linked to expression vectors containingregulatory sequences such as transcription control sequences,translation control sequences, origins of replication, and otherregulatory sequences that are compatible with the host cell and thatcontrol the expression of nucleic acid molecules of the presentinvention. In particular, recombinant molecules of the present inventioninclude nucleic acid molecules that are operatively linked to one ormore expression control sequences. The phrase “operatively linked”refers to linking a nucleic acid molecule to an expression controlsequence in a manner such that the molecule is expressed whentransfected (i.e., transformed, transduced or transfected) into a hostcell.

According to the present invention, the term “transfection” is used torefer to any method by which an exogenous nucleic acid molecule (i.e., arecombinant nucleic acid molecule) can be inserted into a cell. The term“transformation” can be used interchangeably with the term“transfection” when such term is used to refer to the introduction ofnucleic acid molecules into microbial cells, such as algae, bacteria andyeast. In microbial systems, the term “transformation” is used todescribe an inherited change due to the acquisition of exogenous nucleicacids by the microorganism and is essentially synonymous with the term“transfection.” Therefore, transfection techniques include, but are notlimited to, transformation, chemical treatment of cells, particlebombardment, electroporation, microinjection, lipofection, adsorption,infection and protoplast fusion.

The following experimental results are provided for purposes ofillustration and are not intended to limit the scope of the invention.

EXAMPLES Example 1

The following example describes the production of a yeast-basedimmunotherapeutic composition for the treatment or prevention ofhepatitis B virus (HBV) infection.

In this experiment, yeast (e.g., Saccharomyces cerevisiae) wereengineered to express various HBV surface-core fusion proteins, eachhaving the basic structure shown in FIG. 2, under the control of thecopper-inducible promoter, CUP1, or the TEF2 promoter. In each case, theHBV fusion protein was a single polypeptide of approximately 595 aminoacids, with the following sequence elements fused in frame from N- toC-terminus, represented by SEQ ID NO:34 (1) an N-terminal peptide toimpart resistance to proteasomal degradation and stabilize expression(positions 1 to 6 of SEQ ID NO:34); 2) a two amino acid spacer (Thr-Ser)to introduce a SpeI restriction enzyme site; 3) the amino acid sequenceof a near full-length (minus position 1) HBV genotype C large (L)surface antigen (e.g., positions 9 to 407 of SEQ ID NO:34, correspondingto positions 2-400 of SEQ ID NO:11, which differs from SEQ ID NO:34 atpositions 350-351 of SEQ ID NO:11, where a Leu-Val sequence in SEQ IDNO:11 is replaced with a Gln-Ala sequence at positions 357-358 of SEQ IDNO:34); 4) the amino acid sequence of an HBV core antigen (e.g.,positions 408 to 589 of SEQ ID NO:34 or positions 31-212 of SEQ IDNO:9); and 5) a hexahistidine tag (positions 590-595 of SEQ ID NO:34). Anucleic acid sequence encoding the fusion protein of SEQ ID NO:34 (codonoptimized for yeast expression) is represented herein by SEQ ID NO:33.Positions 28-54 of SEQ ID NO:34 comprise the hepatocyte receptor portionof large (L) surface protein. SEQ ID NO:34 contains multiple epitopes ordomains that are believed to enhance the immunogenicity of the fusionprotein. For example, at positions 209-220, positions 389-397, positions360-367, and positions 499-506, with respect to SEQ ID NO:34, compriseknown MHC Class I binding and/or CTL epitopes. Positions 305-328 of SEQID NO:34 comprise an antibody epitope. This fusion protein andcorresponding yeast-based immunotherapeutic comprising this protein canbe generally referred to herein as “Score”, “MADEAP-Score”, “M-Score”,or “GI-13002”.

Briefly, DNA encoding nearly full length large surface antigen (L) fusedto full length core antigen was codon optimized for expression in yeast,and then digested with EcoRI and NotI and inserted behind the CUP1promoter (pGI-100), or the TEF2 promoter (pTK57-1), in yeast 2 umexpression vectors. The fusion protein encoded by these constructs isrepresented herein by SEQ ID NO:34 (encoded by nucleotide sequence SEQID NO:33) and has an expected approximate molecular weight of 66 kDa.The resulting plasmids were introduced into Saccharomyces cerevisiaeW303α yeast by Lithium acetate/polyethylene glycol transfection, andprimary transfectants were selected on solid minimal plates lackinguracil (UDM; uridine dropout medium). Colonies were re-streaked onto UDMor ULDM (uridine and leucine dropout medium) and allowed to grow for 3days at 30° C. Liquid cultures lacking uridine (U2 medium: 20 g/Lglucose; 6.7 g/L of yeast nitrogen base containing ammonium sulfate;0.04 mg/mL each of histidine, leucine, tryptophan, and adenine) orlacking uridine and leucine (UL2 medium: 20 g/L glucose; 6.7 g/L ofyeast nitrogen base containing ammonium sulfate; and 0.04 mg/mL each ofhis, tryptophan, and adenine) were inoculated from plates and startercultures were grown for 20 h at 30° C., 250 rpm. pH buffered mediacontaining 4.2 g/L of Bis-Tris (BT-U2; BT-UL2) was also inoculated toevaluate growth of the yeast under neutral pH conditions. Primarycultures were used to inoculate final cultures of the same formulationand growth was continued until a density or 1.1 to 4.0 YU/mL wasreached.

For TEF2 strains (constitutive expression), cells were harvested, washedand heat killed at 56° C. for 1 h in PBS. Live cells were also processedfor comparison. For CUP1 strains (inducible expression), expression wasinduced in the same medium with 0.5 mM copper sulfate for 5 h at 30° C.,250 rpm. Cells were harvested, washed and heat killed at 56° C. for 1 hin PBS. Live cells were also processed for comparison.

After heat kill of TEF2 and CUP1 cultures, cells were washed three timesin PBS. Total protein expression was measured by a TCAprecipitation/nitrocellulose binding assay and antigen expression wasmeasured by western blot using an anti-his tag monoclonal antibody. Theantigen was quantified by interpolation from a standard curve ofrecombinant, hexa-histidine tagged NS3 protein that was processed on thesame western blot. Results are shown in FIG. 16 (heat-killed) and FIG.17 (live yeast). These figures show that the yeast-based immunotherapycomposition of the invention expresses the HBV surface-core fusionprotein well using both promoters, and can be identified by Western blotin both heat-killed and live yeast cells. The calculated antigenexpression by this yeast-based immunotherapeutic was ˜5000 ng proteinper Y.U. (Yeast Unit; One Yeast Unit (Y.U.) is 1×10⁷ yeast cells oryeast cell equivalents) or 76 pmol protein per Y.U.

Example 2

The following example describes the production of another yeast-basedimmunotherapeutic composition for the treatment or prevention ofhepatitis B virus (HBV) infection.

Yeast (e.g., Saccharomyces cerevisiae) were engineered to expressvarious HBV fusion proteins, each having the structure schematicallyshown in FIG. 3, under the control of the copper-inducible promoter,CUP1, or the TEF2 promoter. In each case, the fusion protein was asingle polypeptide of approximately 945 amino acids, with the followingsequence elements fused in frame from N- to C-terminus, represented bySEQ ID NO:36: (1) an N-terminal peptide to impart resistance toproteasomal degradation and stabilize expression (positions 1 to 5 ofSEQ ID NO:36); 2) the amino acid sequence of an HBV genotype Chepatocyte receptor domain of the pre-S1 portion of HBV large (L)surface protein (unique to L) (e.g., positions 21-47 of SEQ ID NO:11 orpositions 6 to 32 of SEQ ID NO:36); 3) the amino acid sequence of afull-length HBV genotype C small (S) surface antigen (e.g., positions176 to 400 of SEQ ID NO:11 or positions 33 to 257 of SEQ ID NO:36); 4) atwo amino acid spacer/linker (Leu-Glu) to facilitate cloning andmanipulation of the sequences (positions 258 and 259 of SEQ ID NO:36);5) the amino acid sequence of a portion of the HBV genotype C polymeraseincluding the reverse transcriptase domain (e.g., positions 247 to 691of SEQ ID NO:10 or positions 260 to 604 of SEQ ID NO:36); 6) an HBVgenotype C core protein (e.g., positions 31-212 of SEQ ID NO:9 orpositions 605 to 786 of SEQ ID NO:36); 7) the amino acid sequence of anHBV genotype C X antigen (e.g., positions 2 to 154 of SEQ ID NO:12 orpositions 787 to 939 of SEQ ID NO:36); and 8) a hexahistidine tag(positions 940 to 945 of SEQ ID NO:36). This fusion protein andcorresponding yeast-based immunotherapeutic comprising this protein canbe generally referred to herein as “MADEAP-Spex”, “M-Spex”, or“GI-13005”.

A nucleic acid sequence encoding the fusion protein of SEQ ID NO:36(codon optimized for yeast expression) is represented herein by SEQ IDNO:35. SEQ ID NO:36 has an expected approximate molecular weight of106-107 kDa. SEQ ID NO:36 contains multiple epitopes or domains that arebelieved to enhance the immunogenicity of the fusion protein, includingseveral described above for SEQ ID NO:34. In addition, the reversetranscriptase domain used in this fusion protein contains several aminoacid positions that are known to become mutated as a drug-resistanceresponse to treatment with anti-viral drugs, and therefore, may bemutated in this fusion protein in order to provide a therapeutic orprophylactic immunotherapeutic that targets specific drug resistance(escape) mutations. These amino acid positions are, with respect to SEQID NO:36, at amino acid position: 432 (Val, known to mutate to a Leuafter lamivudine therapy); position 439 (Leu, known to mutate to a Metafter lamivudine therapy); position 453 (Ala, known to mutate to a Thrafter tenofovir therapy); position 463 (Met, known to mutate to an Ileor Val after lamivudine therapy); and position 495 (Asn, known to mutateto Thr after adefovir therapy).

To create a second yeast-based immunotherapeutic utilizing a differentN-terminal peptide in the antigen, yeast (e.g., Saccharomycescerevisiae) were engineered to express various HBV fusion proteins, alsohaving the basic structure schematically shown in FIG. 3, under thecontrol of the copper-inducible promoter, CUP1, or the TEF2 promoter. Inthis second case, an alpha factor prepro sequence (represented by SEQ IDNO:89) was used in place of the synthetic N-terminal peptide describedabove in the fusion represented by SEQ ID NO:36. Briefly, the new fusionprotein was a single polypeptide with the following sequence elementsfused in frame from N- to C-terminus, represented by SEQ ID NO:92: (1)an N-terminal peptide to impart resistance to proteasomal degradationand stabilize or enhance expression (SEQ ID NO:89, positions 1 to 89 ofSEQ ID NO:92); 2) a two amino acid spacer/linker (Thr-Ser) to facilitatecloning and manipulation of the sequences (positions 90 to 91 of SEQ IDNO:92); 3) the amino acid sequence of an HBV genotype C hepatocytereceptor domain of the pre-S1 portion of HBV large (L) surface protein(unique to L) (e.g., positions 21-47 of SEQ ID NO:11 or positions 92 to118 of SEQ ID NO:92); 4) the amino acid sequence of a full-length HBVgenotype C small (S) surface antigen (e.g., positions 176 to 400 of SEQID NO:11 or positions 119 to 343 of SEQ ID NO:92); 5) a two amino acidspacer/linker (Leu-Glu) to facilitate cloning and manipulation of thesequences (e.g., positions 344 to 345 of SEQ ID NO:92); 6) the aminoacid sequence of a portion of the HBV genotype C polymerase includingthe reverse transcriptase domain (e.g., positions 247 to 691 of SEQ IDNO:10 or positions 346 to 690 of SEQ ID NO:92); 7) an HBV genotype Ccore protein (e.g., positions 31-212 of SEQ ID NO:9 or positions 691 to872 of SEQ ID NO:92); 8) the amino acid sequence of an HBV genotype C Xantigen (e.g., positions 2 to 154 of SEQ ID NO:12 or positions 873 to1025 of SEQ ID NO:92); and 9) a hexahistidine tag (e.g., positions 1026to 1031 of SEQ ID NO:92). This fusion protein and correspondingyeast-based immunotherapeutic comprising this protein can be generallyreferred to herein as “alpha-Spex”, “a-Spex”, or GI-13004”.

A nucleic acid sequence encoding the fusion protein of SEQ ID NO:92(codon optimized for yeast expression) is represented herein by SEQ IDNO:91. SEQ ID NO:92 has an expected approximate molecular weight of 123kDa. SEQ ID NO:92 contains multiple epitopes or domains that arebelieved to enhance the immunogenicity of the fusion protein, includingseveral described above for SEQ ID NO:34 and SEQ ID NO:36. In addition,the reverse transcriptase domain used in this fusion protein containsseveral amino acid positions that are known to become mutated as adrug-resistance response to treatment with anti-viral drugs, andtherefore, may be mutated in this fusion protein in order to provide atherapeutic or prophylactic immunotherapeutic that targets specific drugresistance (escape) mutations. These amino acid positions are, withrespect to SEQ ID NO:92, at amino acid position: 518 (Val, known tomutate to a Leu after lamivudine therapy); position 525 (Leu, known tomutate to a Met after lamivudine therapy); position 539 (Ala, known tomutate to a Thr after tenofovir therapy); position 549 (Met, known tomutate to an Ile or Val after lamivudine therapy); and position 581(Asn, known to mutate to Thr after adefovir therapy).

To create these immunotherapeutic compositions comprising the amino acidsequences represented by SEQ ID NO:36 and SEQ ID NO:92, DNA encoding theabove-described conserved regions of surface antigen (hepatocytereceptor region of pre-S1 or large surface antigen, and full-lengthsmall surface antigen) and the reverse transcriptase region ofpolymerase were fused to full length core and full length X antigen. TheDNA was codon-optimized for expression in yeast and then digested withEcoRI and NotI and inserted behind the CUP1 promoter (pGI-100) or theTEF2 promoter (pTK57-1) in yeast 2 um expression vectors. The resultingplasmids were introduced into Saccharomyces cerevisiae W303α yeast byLithium acetate/polyethylene glycol transfection, and primarytransfectants were selected on solid minimal plates lacking Uracil (UDM;uridine dropout medium). Colonies were re-streaked onto UDM or ULDM(uridine and leucine dropout medium) and allowed to grow for 3 days at30° C.

Liquid cultures lacking uridine (U2) or lacking uridine and leucine(UL2) were inoculated from plates and starter cultures were grown for 20h at 30° C., 250 rpm. pH buffered Media containing 4.2 g/L of Bis-Tris(BT-U2; BT-UL2) were also inoculated to evaluate growth of the yeastunder neutral pH conditions (data not shown). Primary cultures were usedto inoculate final cultures of the same formulation and growth wascontinued until a density or 1.1 to 4.0 YU/mL was reached. For TEF2strains (constitutive expression), cells were harvested, washed and heatkilled at 56° C. for 1 h in PBS. For CUP1 strains (inducibleexpression), expression was induced in the same medium with 0.5 mMcopper sulfate for 5 h at 30° C., 250 rpm. Cells were harvested, washedand heat killed at 56° C. for 1 h in PBS. Live cells were also processedfor comparison (data not shown).

After heat kill of TEF2 and CUP1 cultures, cells were washed three timesin PBS. Total protein expression was measured by a TCAprecipitation/nitrocellulose binding assay and antigen expression wasmeasured by western blot using an anti-his tag monoclonal antibody. Theantigen was quantified by interpolation from a standard curve ofrecombinant, hexa-histidine tagged NS3 protein that was processed on thesame western blot.

For the yeast-based immunotherapeutic expressing the fusion proteinrepresented by SEQ ID NO:36 (GI-13005), results are shown in FIG. 18.FIG. 18 shows that the yeast-based immunotherapy composition of theinvention expresses the fusion protein well using both promoters, andcan be identified by Western blot in heat-killed yeast cells (expressionwas also achieved in live yeast cells, data not shown). The calculatedantigen expression by this yeast-based immunotherapeutic was ˜1200 ngprotein per Y.U. or 11 pmol protein per Y.U., for growth in UL2.

For the yeast-based immunotherapeutic expressing the fusion proteinrepresented by SEQ ID NO:92 (GI-13004), results are shown in FIG. 19.FIG. 19 shows expression of this yeast-based immunotherapy compositionunder the control of the CUP1 promoter (identified in FIG. 19 asAlpha-SPEX) as compared to a yeast-based immunotherapeutic thatexpresses an unrelated antigen (Control Yeast) and to the yeast-basedimmunotherapeutic composition expressing an HBV fusion proteinrepresented by SEQ ID NO:36 (SPEX). FIG. 19 shows that the yeast-basedimmunotherapeutics expresses the relevant fusion proteins well, and canbe identified by Western blot in heat-killed yeast cells. The calculatedantigen expression by this yeast-based immunotherapeutic (Alpha-SPEX)was ˜5000 ng protein per Y.U. or 41 pmol protein per Y.U. for growth inUL2.

Example 3

The following example describes the production of additional yeast-basedimmunotherapeutic composition for the treatment or prevention ofhepatitis B virus (HBV) infection.

In this experiment, yeast (e.g., Saccharomyces cerevisiae) areengineered to express various HBV polymerase-core fusion proteins, asshown schematically in FIG. 4, under the control of the copper-induciblepromoter, CUP1, or the TEF2 promoter. In each case, the fusion proteinis a single polypeptide of approximately 527 amino acids, with thefollowing sequence elements fused in frame from N- to C-terminus,represented by SEQ ID NO:38: (1) an N-terminal peptide to impartresistance to proteasomal degradation and stabilize expression (SEQ IDNO:37; positions 1 to 6 of SEQ ID NO:38); 2) the amino acid sequence ofa portion of the HBV genotype C polymerase including the reversetranscriptase domain (e.g., positions 347 to 691 of SEQ ID NO:10 orpositions 7 to 351 of SEQ ID NO:38); 3) an HBV genotype C core protein(e.g., positions 31 to 212 of SEQ ID NO:9 or positions 352 to 533 of SEQID NO:38); and 4) a hexahistidine tag (e.g., positions 534 to 539 of SEQID NO:38). SEQ ID NO:38 has a predicted molecular weight ofapproximately 58 kDa. The sequence also contains epitopes or domainsthat are believed to enhance the immunogenicity of the fusion protein.In additional constructs, the N-terminal peptide of SEQ ID NO:37 isreplaced with a different synthetic N-terminal peptide represented by ahomologue of SEQ ID NO:37 that meets the same basic structuralrequirements of SEQ ID NO:37 as described in detail in thespecification, or the N-terminal peptide of SEQ ID NO:37 is replacedwith the N-terminal peptide of SEQ ID NO:89 or SEQ ID NO:90, and inanother construct, the N-terminal peptide is omitted and a methionine isincluded at position one.

In another experiment, yeast (e.g., Saccharomyces cerevisiae) areengineered to express various HBV X-core fusion proteins as shownschematically in FIG. 5 under the control of the copper-induciblepromoter, CUP1, or the TEF2 promoter. In each case, the fusion proteinis a single polypeptide of approximately 337 amino acids with thefollowing sequence elements fused in frame from N- to C-terminus,represented by SEQ ID NO:39 (1) an N-terminal peptide to impartresistance to proteasomal degradation and stabilize expression (SEQ IDNO:37; positions 1 to 6 of SEQ ID NO:39); 2) the amino acid sequence ofa near full-length (minus position 1) HBV genotype C X antigen (e.g.,positions 2 to 154 of SEQ ID NO:12 or positions 7 to 159 of SEQ IDNO:39); 3) an HBV genotype C core protein (e.g., positions 31 to 212 ofSEQ ID NO:9 or positions 160 to 341 of SEQ ID NO:39); and 4) ahexahistidine tag (positions 342 to 347 of SEQ ID NO:39). SEQ ID NO:39has a predicted approximate molecular weight of 37 kDa. The sequencealso contains epitopes or domains that are believed to enhance theimmunogenicity of the fusion protein. In additional constructs, theN-terminal peptide of SEQ ID NO:37 is replaced with a differentsynthetic N-terminal peptide represented by a homologue of SEQ ID NO:37that meets the same basic structural requirements of SEQ ID NO:37 asdescribed in detail in the specification, or the N-terminal peptide ofSEQ ID NO:37 is replaced with the N-terminal peptide of SEQ ID NO:89 orSEQ ID NO:90, and in another construct, the N-terminal peptide isomitted and a methionine is included at position one.

In another experiment, yeast (e.g., Saccharomyces cerevisiae) areengineered to express various HBV polymerase proteins as shownschematically in FIG. 6 under the control of the copper-induciblepromoter, CUP1, or the TEF2 promoter. In each case, the fusion proteinis a single polypeptide with the following sequence elements fused inframe from N- to C-terminus, represented by SEQ ID NO:40 (1) anN-terminal peptide to impart resistance to proteasomal degradation andstabilize expression (SEQ ID NO:37, or positions 1 to 6 of SEQ ID NO:40;2) the amino acid sequence of a portion of the HBV genotype C polymeraseincluding the reverse transcriptase domain (e.g., positions 347 to 691of SEQ ID NO:10 or positions 7 to 351 of SEQ ID NO:40); and 3) ahexahistidine tag (positions 352 to 357 of SEQ ID NO:40). The sequencealso contains epitopes or domains that are believed to enhance theimmunogenicity of the fusion protein. In addition, in one embodiment,the sequence of this construct can be modified to introduce one or moreor all of the following anti-viral resistance mutations: rtM2041,rtL180M, rtM204V, rtV173L, rtN236T, rtA194T (positions given withrespect to the full-length amino acid sequence for HBV polymerase). Inone embodiment, six different immunotherapy compositions are created,each one containing one of these mutations. In other embodiments, all orsome of the mutations are included in a single fusion protein. Inadditional constructs, the N-terminal peptide of SEQ ID NO:37 isreplaced with a different synthetic N-terminal peptide represented by ahomologue of SEQ ID NO:37 that meets the same basic structuralrequirements of SEQ ID NO:37 as described in detail in thespecification, or the N-terminal peptide of SEQ ID NO:37 is replacedwith the N-terminal peptide of SEQ ID NO:89 or SEQ ID NO:90, and inanother construct, the N-terminal peptide is omitted and a methionine isincluded at position one.

In another experiment, yeast (e.g., Saccharomyces cerevisiae) areengineered to express various HBV polymerase-surface-core fusionproteins as shown schematically in FIG. 7 under the control of thecopper-inducible promoter, CUP1, or the TEF2 promoter. In each case, thefusion protein is a single polypeptide with the following sequenceelements fused in frame from N- to C-terminus, represented by SEQ IDNO:41: (1) an N-terminal peptide to impart resistance to proteasomaldegradation and stabilize expression (e.g., positions 1 to 5 of SEQ IDNO:41); 2) an amino acid sequence of the amino HBV hepatocyte receptordomain of the pre-S1 portion of HBV large (L) surface protein (unique toL) (e.g., positions 21-47 of SEQ ID NO:11 or positions 6 to 32 of SEQ IDNO:41); 3) the amino acid sequence of an HBV small (S) surface protein(e.g., positions 176 to 400 of SEQ ID NO:11 or positions 33 to 257 ofSEQ ID NO:41); 4) a two amino acid spacer/linker to facilitate cloningand manipulation of the sequences (e.g., positions 258 and 259 of SEQ IDNO:41); 5) the amino acid sequence of an HBV polymerase comprising thereverse transcriptase domain (e.g., positions 247 to 691 of SEQ ID NO:10or positions 260 to 604 of SEQ ID NO:41); 6) the amino acid sequence ofan HBV core protein (e.g., positions 31-212 of SEQ ID NO:9 or positions605 to 786 of SEQ ID NO:41); and 7) a hexahistidine tag (e.g., positions787 to 792 of SEQ ID NO:41). The sequence also contains epitopes ordomains that are believed to enhance the immunogenicity of the fusionprotein. In addition, in one embodiment, the sequence of this constructcan be modified to introduce one or more or all of the followinganti-viral resistance mutations: rtM2041, rtL180M, rtM204V, rtV173L,rtN236T, rtA194T (positions given with respect to the full-length aminoacid sequence for HBV polymerase). In one embodiment, six differentimmunotherapy compositions are created, each one containing one of thesemutations. In other embodiments, all or some of the mutations areincluded in a single fusion protein. In one embodiment, this constructalso contains one or more anti-viral resistance mutations in the surfaceantigen. In additional constructs, the N-terminal peptide represented bypositions 1 to 5 of SEQ ID NO:41 is replaced with a different syntheticN-terminal peptide represented by a homologue of positions 1 to 5 of SEQID NO:41 that meets the same basic structural requirements of positions1 to 5 of SEQ ID NO:41 (or of SEQ ID NO:37) as described in detail inthe specification, or the N-terminal peptide of positions 1 to 5 of SEQID NO:41 is replaced with the N-terminal peptide of SEQ ID NO:89 or SEQID NO:90, and in another construct, the N-terminal peptide is omittedand a methionine is included at position one.

To produce any of the above-described fusion proteins and yeast-basedimmunotherapy compositions expressing such proteins, briefly, DNAencoding the fusion protein is codon optimized for expression in yeastand then digested with EcoRI and NotI and inserted behind the CUP1promoter (pGI-100) or the TEF2 promoter (pTK57-1) in yeast 2 umexpression vectors. The resulting plasmids are introduced intoSaccharomyces cerevisiae W303α yeast by Lithium acetate/polyethyleneglycol transfection, and primary transfectants are selected on solidminimal plates lacking Uracil (UDM; uridine dropout medium). Coloniesare re-streaked onto UDM or ULDM (uridine and leucine dropout medium)and allowed to grow for 3 days at 30° C.

Liquid cultures lacking uridine (U2) or lacking uridine and leucine(UL2) are inoculated from plates and starter cultures were grown for 20h at 30° C., 250 rpm. pH buffered Media containing 4.2 g/L of Bis-Tris(BT-U2; BT-UL2) can also be inoculated to evaluate growth of the yeastunder neutral pH conditions. Primary cultures are used to inoculatefinal cultures of the same formulation and growth is continued until adensity or 1.1 to 4.0 YU/mL is reached. For TEF2 strains (constitutiveexpression), cells are harvested, washed and heat killed at 56° C. for 1h in PBS. For CUP1 strains (inducible expression), expression is inducedin the same medium with 0.5 mM copper sulfate for 5 h at 30° C., 250rpm. Cells are harvested, washed and heat killed at 56° C. for 1 h inPBS. Live cells are also processed for comparison.

After heat kill of TEF2 and CUP1 cultures, cells are washed three timesin PBS. Total protein expression is measured by a TCAprecipitation/nitrocellulose binding assay and protein expression ismeasured by western blot using an anti-his tag monoclonal antibody.Fusion protein is quantified by interpolation from a standard curve ofrecombinant, hexa-histidine tagged NS3 protein that was processed on thesame western blot.

Example 4

The following example describes the production of additional yeast-basedimmunotherapeutic compositions for the treatment or prevention ofhepatitis B virus (HBV) infection.

This example describes the production of four different yeast-basedimmunotherapeutic compositions, each one designed to express one HBVprotein. These “single HBV protein yeast immunotherapeutics” can be usedin combination or in sequence with each other and/or in combination orin sequence with other yeast-based immunotherapeutics, such as thosedescribed in any of Examples 1-3 and 5-8, including multi-HBV proteinyeast-based immunotherapeutics described herein. In addition, a “singleHBV protein yeast immunotherapeutic”, such as those described in thisexample, can be produced using the HBV sequence for any given genotypeor sub-genotype, and additional HBV surface antigen yeast-basedimmunotherapeutics can be produced using the HBV sequences for any oneor more additional genotypes or sub-genotypes, in order to provide a“spice rack” of different HBV antigens and genotypes and/orsubgenotypes, each of which is provided in the context of a yeast-basedimmunotherapeutic of the invention, or in an immunization/administrationstrategy that includes at least one yeast-based immunotherapeutic of theinvention.

In this example, the following four yeast-based immunotherapeuticproducts are produced:

HBV Surface Antigen.

Saccharomyces cerevisiae are engineered to express an HBV surfaceprotein under the control of the copper-inducible promoter, CUP1, or theTEF2 promoter. In each case, the fusion protein is a single polypeptidewith the following sequence elements fused in frame from N- toC-terminus, represented by SEQ ID NO:93: 1) an N-terminal peptide of SEQID NO:89 (positions 1-89 of SEQ ID NO:93); 2) the amino acid sequence ofa near full-length (minus position 1) HBV genotype C large (L) surfaceantigen (e.g., positions 2-400 of SEQ ID NO:11 or positions 90 to 488 ofSEQ ID NO:93); and 3) a hexahistidine tag (e.g., positions 489 to 494 ofSEQ ID NO:93). Alternatively, the N-terminal peptide can be replacedwith SEQ ID NO:37 or a homologue thereof or another N-terminal peptidedescribed herein.

HBV Polymerase Antigen.

Saccharomyces cerevisiae are engineered to express the following HBVPolymerase protein under the control of the copper-inducible promoter,CUP1, or the TEF2 promoter. In each case, the fusion protein is a singlepolypeptide with the following sequence elements fused in frame from N-to C-terminus, represented by SEQ ID NO:94: 1) an N-terminal peptide ofSEQ ID NO:89 (positions 1-89 of SEQ ID NO:94); 2) the amino acidsequence of a portion of the HBV genotype C polymerase including thereverse transcriptase domain (e.g., positions 347 to 691 of SEQ ID NO:10or positions 90 to 434 of SEQ ID NO:94); and 3) a hexahistidine tag(e.g., positions 435 to 440 of SEQ ID NO:94). Alternatively, theN-terminal peptide can be replaced with SEQ ID NO:37 or a homologuethereof or another N-terminal peptide described herein.

HBV Core Antigen.

Saccharomyces cerevisiae are engineered to express the following HBVCore protein under the control of the copper-inducible promoter, CUP1,or the TEF2 promoter. In each case, the fusion protein is a singlepolypeptide with the following sequence elements fused in frame from N-to C-terminus, represented by SEQ ID NO:95: 1) an N-terminal peptide ofSEQ ID NO:89 (positions 1-89 of SEQ ID NO:95); 2) the amino acidsequence of a portion of the HBV genotype C Core protein (e.g.,positions 31 to 212 of SEQ ID NO:9 or positions 90 to 271 of SEQ IDNO:95); and 3) a hexahistidine tag (e.g., positions 272 to 277 of SEQ IDNO:95). Alternatively, the N-terminal peptide can be replaced with SEQID NO:37 or a homologue thereof or another N-terminal peptide describedherein.

HBV X Antigen.

Saccharomyces cerevisiae are engineered to express the following HBV Xantigen under the control of the copper-inducible promoter, CUP1, or theTEF2 promoter. In each case, the fusion protein is a single polypeptidewith the following sequence elements fused in frame from N- toC-terminus, represented by SEQ ID NO:96: 1) an N-terminal peptide of SEQID NO:89 (positions 1-89 of SEQ ID NO:96); 2) the amino acid sequence ofa portion of the HBV genotype C X antigen (e.g., positions 2 to 154 ofSEQ ID NO:12 or positions 90 to 242 of SEQ ID NO:96); and 3) ahexahistidine tag (e.g., positions 243 to 248 of SEQ ID NO:96).Alternatively, the N-terminal peptide can be replaced with SEQ ID NO:37or a homologue thereof or another N-terminal peptide described herein.

To create these immunotherapeutic compositions, briefly, DNA encodingthe fusion protein is codon optimized for expression in yeast and thendigested with EcoRI and NotI and inserted behind the CUP1 promoter(pGI-100) or the TEF2 promoter (pTK57-1) in yeast 2 um expressionvectors. The resulting plasmids are introduced into Saccharomycescerevisiae W303α yeast by Lithium acetate/polyethylene glycoltransfection, and primary transfectants are selected on solid minimalplates lacking uracil (UDM; uridine dropout medium). Colonies arere-streaked onto UDM or ULDM (uridine and leucine dropout medium) andallowed to grow for 3 days at 30° C.

Liquid cultures lacking uridine (U2) or lacking uridine and leucine(UL2) are inoculated from plates and starter cultures were grown for 20h at 30° C., 250 rpm. pH buffered Media containing 4.2 g/L of Bis-Tris(BT-U2; BT-UL2) may also be inoculated to evaluate growth of the yeastunder neutral pH conditions. Primary cultures are used to inoculatefinal cultures of the same formulation and growth is continued until adensity or 1.1 to 4.0 YU/mL is reached. For TEF2 strains (constitutiveexpression), cells are harvested, washed and heat killed at 56° C. for 1h in PBS. For CUP1 strains (inducible expression), expression is inducedin the same medium with 0.5 mM copper sulfate for 5 h at 30° C., 250rpm. Cells are harvested, washed and heat killed at 56° C. for 1 h inPBS. Live cells are also processed for comparison.

After heat kill of TEF2 and CUP1 cultures, cells are washed three timesin PBS. Total protein expression is measured by a TCAprecipitation/nitrocellulose binding assay and protein expression ismeasured by western blot using an anti-his tag monoclonal antibody.Fusion protein is quantified by interpolation from a standard curve ofrecombinant, hexa-histidine tagged NS3 protein that was processed on thesame western blot.

Example 5

The following example describes the production of several differentyeast-based immunotherapeutic compositions for the treatment orprevention of hepatitis B virus (HBV) infection.

This example describes the production of yeast-based immunotherapeuticsexpressing proteins that have been designed to achieve one or more ofthe following goals: (1) produce a multi-antigen HBV construct thatcomprises less than about 690 amino acids (corresponding to less thantwo thirds of the HBV genome), in order to produce a yeast-basedimmunotherapeutic clinical product that is compliant with the guidelinesof the Recombinant DNA Advisory Committee (RAC), if necessary; (2)produce a multi-antigen HBV construct containing a maximized number ofknown T cell epitopes associated with immune responses toacute/self-limiting HBV infections and/or chronic HBV infections; (3)produce a multi-antigen HBV construct containing T cell epitopes thatare most conserved among genotypes; and/or (4) produce a multi-antigenHBV construct modified to correspond more closely to one or moreconsensus sequences, consensus epitopes, and/or epitope(s) fromparticular genotypes. The modifications demonstrated in this example canbe applied individually or together to any other yeast-basedimmunotherapeutic described or contemplated herein.

In one experiment, a yeast-based immunotherapeutic composition thatcomprises a yeast expressing a fusion protein meeting the requirementsof the goals specified above, and comprising portions of each of the HBVmajor proteins: HBV surface antigen, polymerase, core and X antigen, wasdesigned. To design this fusion protein, individual HBV antigens withinthe fusion were reduced in size (as compared to full-length), and thefusion segments were individually modified to maximize the inclusion ofknown T cell epitopes corresponding to those identified in Table 5.Inclusion of T cell epitopes in this fusion protein was prioritized asfollows:

-   -   Epitopes identified in immune responses to both        acute/self-limiting HBV infections and chronic HBV        infections>Epitopes identified in immune responses to        acute/self-limiting HBV infections>Epitopes identified in immune        responses to chronic HBV infections

Artificial junctions were also minimized in the design of each segmentof this fusion protein because, without being bound by theory, it isbelieved that natural evolution has resulted in: i) contiguous sequencesin the virus that express well; and ii) an immunoproteasome in antigenpresenting cells that can properly digest and present those sequences tothe immune system. Accordingly, a fusion protein with many unnaturaljunctions may be less useful in a yeast-based immunotherapeutic ascompared to one that retains more of the natural HBV protein sequences.

To construct a segment comprising HBV surface antigen for use in afusion protein, a full-length large (L) surface antigen protein from HBVgenotype C was reduced in size by truncation of the N- and C-terminalsequences (positions 1 to 119 and positions 369 to 400 of large antigenwere removed, as compared to a full-length L surface antigen protein,such as that represented by SEQ ID NO:11). The remaining portion wasselected, in part, to maximize the inclusion of known MHC Class I T cellepitopes corresponding to those identified in Table 5, using theprioritization for inclusion of T cell epitopes described above. Theresulting surface antigen segment is represented by SEQ ID NO:97.

To construct the segment comprising HBV polymerase for use in a fusionprotein, substantial portions of a full-length polymerase from HBVgenotype C, which is a very large protein of about 842 amino acids, wereeliminated by focusing on inclusion of the active site domain (from theRT domain), which is the most conserved region of the protein among HBVgenotypes and isolates. The RT domain also includes several sites wheredrug resistance mutations have been known to occur; thus, this portionof the construct can be further modified in other versions, as needed,to target escape mutations of targeted therapy. In fusion proteinsincluding fewer HBV proteins, the size of the polymerase segment can beexpanded, if desired. The selected portion of the HBV polymerase wasincluded to maximize known T cell epitopes, using the prioritizationstrategy discussed above. Sequence of full-length polymerase that wastherefore eliminated included sequence outside of the RT domain, andsequences within the RT domain that contained no known T cell epitopes,or that included two epitopes identified in less than 17% or 5%,respectively, of genotype A patients where these epitopes wereidentified (see Desmond et al., 2008 and Table 5). All but one of theremaining T cell epitopes in the HBV polymerase genotype C segment wereperfect matches to the published epitopes from the genotype A analysis,and the one epitope with a single amino acid mismatch was modified tocorrespond to the published epitope. The resulting HBV polymeraseantigen segment is represented by SEQ ID NO:98.

To construct the segment comprising HBV core antigen for use in a fusionprotein, a full-length Core protein (e.g., similar to positions 31-212of SEQ ID NO:9) from HBV genotype C was modified as follows: i) a singleamino acid within a T cell epitope of the genotype C-derived protein wasmodified to create a perfect match to a known T cell epitope describedin Table 5; ii) seven amino acids of the N-terminus, which did notcontain a T cell epitope, were removed, preserving some flanking aminoacids N-terminal to the first known T cell epitope in the protein; andiii) the 24 C terminal amino acids of Core were removed, which does notdelete known epitopes, but which does remove an exceptionally positivelycharged C-terminus A positively charged C-terminus is a good candidatefor removal from an antigen to be expressed in yeast, as such sequencesmay, in some constructs, be toxic to yeast by competitive interferencewith natural yeast RNA binding proteins which often are arginine rich(positively charged). Accordingly, removal of this portion of Core isacceptable. The resulting HBV Core antigen segment is represented by SEQID NO:99.

To construct a segment comprising HBV X antigen for use in a fusionprotein, a full-length X antigen from HBV genotype C (e.g., similar toSEQ ID NO:12) was truncated at the N- and C-terminus to produce asegment of X antigen that includes most of the known T cell epitopesfrom Table 5, which are clustered in the X antigen. Two of the epitopeswere modified by single amino acid changes to correspond to thepublished T cell epitope sequences, and sequence flanking the T cellepitopes at the ends of the segment was retained to facilitate efficientprocessing and presentation of the correct epitopes by an antigenpresenting cell. The resulting HBV X antigen segment is represented bySEQ ID NO:100.

To construct a complete fusion protein containing all four HBV proteinsegments, the four HBV segments described above were linked(surface-pol-core-X) to form a single protein that optimizes theinclusion of T cell epitopes spanning all proteins encoded by the HBVgenome, and that is expected to meet criteria for viral proteins foranticipated clinical use.

Two different fusion proteins were ultimately created, each with adifferent N-terminal peptide added to enhance and/or stabilizeexpression of the fusion protein in yeast. In addition, a hexahistidinepeptide was included at the C-terminus to aid with the identification ofthe protein. As for all of the other proteins used in the yeast-basedimmunotherapeutic compositions described herein, in additionalconstructs, the N-terminal peptide of SEQ ID NO:37 or SEQ ID NO:89utilized in this example can be replaced with a different syntheticN-terminal peptide (e.g., a homologue of SEQ ID NO:37 that meets thesame basic structural requirements of SEQ ID NO:37 as described indetail in the specification), or with a homologue of the N-terminalpeptide of SEQ ID NO:89 or SEQ ID NO:90, and in another construct, theN-terminal peptide is omitted and a methionine is included at positionone. In addition, linker sequences of one, two, three or more aminoacids may be added between segments of the fusion protein, if desired.Also, while these constructs were designed using HBV proteins fromgenotype C as the backbone, any other HBV genotype, sub-genotype, or HBVproteins from different strains or isolates can be used to design theseprotein segments, as exemplified in Example 7. Finally, if one or moresegments are excluded from the fusion protein as described herein, thenthe sequence from the remaining segments can be expanded to includeadditional T cell epitopes and flanking regions of the proteins (e.g.,see Example 8).

To produce yeast-based immunotherapeutic compositions comprising afusion protein constructed of the HBV segments described above, yeast(e.g., Saccharomyces cerevisiae) are engineered to express various HBVsurface-polymerase-core-X fusion proteins, optimized as discussed above,under the control of the copper-inducible promoter, CUP1, or the TEF2promoter.

In one construct, the fusion protein is a single polypeptide with thefollowing sequence elements fused in frame from N- to C-terminus,represented by SEQ ID NO:101: (1) an N-terminal peptide that is an alphafactor prepro sequence, to impart resistance to proteasomal degradationand stabilize expression represented by SEQ ID NO:89 (positions 1-89 ofSEQ ID NO:101); (2) an optimized portion of an HBV large (L) surfaceantigen represented by SEQ ID NO:97 (positions 90 to 338 of SEQ IDNO:101); (3) an optimized portion of the reverse transcriptase (RT)domain of HBV polymerase represented by SEQ ID NO:98 (positions 339 to566 of SEQ ID NO:101); (4) an optimized portion of HBV Core proteinrepresented by SEQ ID NO:99 (positions 567 to 718 of SEQ ID NO:101); (5)an optimized portion of HBV X antigen represented by SEQ ID NO:100(positions 719 to 778 of SEQ ID NO:101); and (6) a hexahistidine tag(e.g., positions 779 to 784 of SEQ ID NO:101).

In a second construct, the fusion protein is a single polypeptide withthe following sequence elements fused in frame from N- to C-terminus,represented by SEQ ID NO:102: (1) an N-terminal peptide that is asynthetic N-terminal peptide designed to impart resistance toproteasomal degradation and stabilize expression represented by SEQ IDNO:37 (positions 1-6 of SEQ ID NO:102); (2) an optimized portion of anHBV large (L) surface antigen represented by positions 2 to 248 of SEQID NO:97 (positions 7 to 254 of SEQ ID NO:102); (3) an optimized portionof the reverse transcriptase (RT) domain of HBV polymerase representedby SEQ ID NO:98 (positions 255 to 482 of SEQ ID NO:102); (4) anoptimized portion of HBV Core protein represented by SEQ ID NO:99(positions 483 to 634 of SEQ ID NO:102); (5) an optimized portion of HBVX antigen represented by SEQ ID NO:100 (positions 635 to 694 of SEQ IDNO:102); and (6) a hexahistidine tag (e.g., positions 695 to 700 of SEQID NO:102).

Yeast-based immunotherapy compositions expressing these fusion proteinsare produced using the same protocol described in detail in Example 1-4.

Example 6

The following example describes the production of additional yeast-basedHBV immunotherapeutic compositions that maximize the targeting of HBVgenotypes and/or sub-genotypes in conjunction with conserved antigenand/or epitope inclusion within a single composition, in order toprovide single compositions with the potential to treat a large numberof individuals or populations of individuals.

To prepare a construct comprising multiple different genotypes withinthe same yeast-based immunotherapeutic, yeast (e.g., Saccharomycescerevisiae) are engineered to express an HBV fusion protein under thecontrol of a suitable promoter, such as the copper-inducible promoter,CUP1, or the TEF2 promoter. The protein is a single polypeptidecomprising four Core antigens, each one from a different genotype (HBVgenotypes A, B, C and D), represented by SEQ ID NO:105: 1) an N-terminalmethionine at position 1 of SEQ ID NO:105; 2) the amino acid sequence ofa near full-length Core protein from HBV genotype A (e.g., positions 31to 212 of SEQ ID NO:1 or positions 2 to 183 of SEQ ID NO: 105); 3) theamino acid sequence of a near full-length Core protein from HBV genotypeB (e.g., positions 30 to 212 of SEQ ID NO:5 or positions 184 to 395 ofSEQ ID NO: 105); 4) the amino acid sequence of a near full-length Coreprotein from HBV genotype C (e.g., positions 30 to 212 of SEQ ID NO:9 orpositions 396 to 578 of SEQ ID NO: 105); 5) the amino acid sequence of anear full-length Core protein from HBV genotype D (e.g., positions 30 to212 of SEQ ID NO:13 or positions 579 to 761 of SEQ ID NO: 105); and 5) ahexahistidine tag (e.g., positions 762 to 767 of SEQ ID NO: 105). Thesequence also contains epitopes or domains that are believed to enhancethe immunogenicity of the fusion protein. The N-terminal methionine atposition 1 can be substituted with SEQ ID NO:37 or a homologue thereof,or with an alpha prepro sequence of SEQ ID NO:89 or SEQ ID NO:90, or ahomologue thereof, or any other suitable N-terminal sequence if desired.In addition, linker sequences can be inserted between HBV proteins tofacilitate cloning and manipulation of the construct, if desired. Thisis an exemplary construct, as any other combination of HBV genotypesand/or subgenotypes can be substituted into this design as desired toconstruct a single antigen yeast-based HBV immunotherapeutic productwith broad clinical applicability and efficient design formanufacturing. The amino acid sequence of SEQ ID NO:105 also containsseveral known T cell epitopes, and certain epitopes have been modifiedto correspond to the published sequence for the given epitope, which canbe identified by comparison of the sequence to the epitopes shown inTable 5, for example.

To prepare a construct comprising more than one HBV antigen and morethan one genotype within the same yeast-based immunotherapeutic, yeast(e.g., Saccharomyces cerevisiae) are engineered to express an HBV fusionprotein under the control of a suitable promoter, such as thecopper-inducible promoter, CUP1, or the TEF2 promoter. The protein is asingle polypeptide comprising two Core antigens and two X antigens, eachone of the pair from a different genotype (HBV genotypes A and C),represented by SEQ ID NO:106: 1) an N-terminal methionine at position 1of SEQ ID NO:106; 2) the amino acid sequence of a near full-length Coreprotein from HBV genotype A (e.g., positions 31 to 212 of SEQ ID NO:1 orpositions 2 to 183 of SEQ ID NO:106); 3) the amino acid sequence of afull-length X antigen from HBV genotype A (e.g., positions SEQ ID NO:4or positions 184 to 337 of SEQ ID NO:106); 4) the amino acid sequence ofa near full-length Core protein from HBV genotype C (e.g., positions 30to 212 of SEQ ID NO:9 or positions 338 to 520 of SEQ ID NO:106); 5) theamino acid sequence of a full-length X antigen from HBV genotype C(e.g., SEQ ID NO:8 or positions 521 to 674 of SEQ ID NO:106); and 5) ahexahistidine tag (e.g., positions 675 to 680 of SEQ ID NO:106). Thesequence also contains epitopes or domains that are believed to enhancethe immunogenicity of the fusion protein. The N-terminal methionine atposition 1 can be substituted with SEQ ID NO:37 or a homologue thereof,or with an alpha prepro sequence of SEQ ID NO:89 or SEQ ID NO:90, or ahomologue thereof. The amino acid sequence of SEQ ID NO:106 alsocontains several known T cell epitopes, and certain epitopes have beenmodified to correspond to the published sequence for the given epitope,which can be identified by comparison of the sequence to the epitopesshown in Table 5, for example.

Yeast-based immunotherapy compositions expressing these fusion proteinsare produced using the same protocol described in detail in Example 1-4.

Example 7

The following example describes the production of additional yeast-basedHBV immunotherapeutic compositions that utilize consensus sequences forHBV genotypes, further maximizing the targeting of HBV genotypes and/orsub-genotypes in conjunction with conserved antigen and/or epitopeinclusion, in order to provide compositions with the potential to treata large number of individuals or populations of individuals using onecomposition.

To design several constructs that include HBV segments from each ofsurface protein, core, polymerase, and X antigen, the fusion proteinstructure described in Example 5 for SEQ ID NO:101 and SEQ ID NO:102(and therefore the subparts of these fusion proteins represented by SEQID NO:97 (Surface antigen), SEQ ID NO:98 (Polymerase), SEQ ID NO:99(Core antigen), and SEQ ID NO:100 (X antigen)) was used as a template.With reference to consensus sequences for each of HBV genotype A, B, Cand D that were built from multiple sources of HBV sequences (e.g., Yuand Yuan et al, 2010, for S, Core and X, where consensus sequences weregenerated from 322 HBV sequences, or for Pol (RT), from the StanfordUniversity HIV Drug Resistance Database, HBVseq and HBV Site ReleaseNotes), sequences in the template structure were replaced with consensussequences corresponding to the same positions, unless using theconsensus sequence altered one of the known acute self-limiting T cellsepitopes or one of the known polymerase escape mutation sites, in whichcase, these positions followed the published sequence for these epitopesor mutation sites. Additional antigens could be constructed based solelyon consensus sequences or using other published epitopes as they becomeknown.

A first construct based on a consensus sequence for HBV genotype A wasdesigned as follows. Using SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99 andSEQ ID NO:100, which were designed to reduce the size of the fusionsegments (as compared to full-length), to maximize the inclusion ofknown T cell epitopes corresponding to those identified in Table 5(priority as discussed above), and to minimize artificial junctions, newfusion segments were created based on a consensus sequence for HBVgenotype A. The new surface antigen segment is represented by positions1-249 of SEQ ID NO:107. The new polymerase (RT) segment is representedby positions 250-477 of SEQ ID NO:107. The new Core segment isrepresented by positions 478-629 of SEQ ID NO:107. The new X antigensegment is represented by positions 630-689 of SEQ ID NO:107. Thiscomplete fusion protein is a single polypeptide with the followingsequence elements fused in frame from N- to C-terminus, wherein the HBVsequences are represented by SEQ ID NO:107 (non-HBV sequences denoted as“optional” were not included in the base sequence of SEQ ID NO:107, butwere actually added to the fusion protein described in this example):(1) an optional N-terminal peptide that is a synthetic N-terminalpeptide designed to impart resistance to proteasomal degradation andstabilize expression represented by SEQ ID NO:37; (2) an optional linkerpeptide of Thr-Ser; (3) an optimized portion of an HBV large (L) surfaceantigen represented by positions 1 to 249 of SEQ ID NO:107, which is aconsensus sequence for HBV genotype A utilizing the design strategydiscussed above; (4) an optimized portion of the reverse transcriptase(RT) domain of HBV polymerase represented by positions 250 to 477 of SEQID NO:107, which is a consensus sequence for HBV genotype A utilizingthe design strategy discussed above; (5) an optimized portion of HBVCore protein represented by positions 478 to 629 of SEQ ID NO:107, whichis a consensus sequence for HBV genotype A utilizing the design strategydiscussed above; (6) an optimized portion of HBV X antigen representedby positions 630 to 689 of SEQ ID NO:107, which is a consensus sequencefor HBV genotype A utilizing the design strategy discussed above; and(7) an optional hexahistidine tag (six histidine residues followingposition 689 of SEQ ID NO:107). A yeast-based immunotherapy compositionexpressing this complete fusion protein is also referred to herein asGI-13010. The fusion protein and corresponding yeast-basedimmunotherapeutic can also be referred to herein as “SPEXv2-A” or“Spex-A”.

A second construct based on a consensus sequence for HBV genotype B wasdesigned as follows. Using SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99 andSEQ ID NO:100, which were designed to reduce the size of the fusionsegments (as compared to full-length), to maximize the inclusion ofknown T cell epitopes corresponding to those identified in Table 5(priority as discussed above), and to minimize artificial junctions, newfusion segments were created based on a consensus sequence for HBVgenotype B. The new surface antigen segment is represented by positions1-249 of SEQ ID NO:108. The new polymerase (RT) segment is representedby positions 250-477 of SEQ ID NO:108. The new Core segment isrepresented by positions 478-629 of SEQ ID NO:108. The new X antigensegment is represented by positions 630-689 of SEQ ID NO:108. Thisfusion protein is a single polypeptide with the following sequenceelements fused in frame from N- to C-terminus, represented by SEQ IDNO:108 (non-HBV sequences denoted as “optional” were not included in thebase sequence of SEQ ID NO:108, but were actually added to the fusionprotein described in this example): (1) an optional N-terminal peptidethat is a synthetic N-terminal peptide designed to impart resistance toproteasomal degradation and stabilize expression represented by SEQ IDNO:37; (2) an optional linker peptide of Thr-Ser; (3) an optimizedportion of an HBV large (L) surface antigen represented by positions 1to 249 of SEQ ID NO:108, which is a consensus sequence for HBV genotypeB utilizing the design strategy discussed above; (4) an optimizedportion of the reverse transcriptase (RT) domain of HBV polymeraserepresented by positions 250 to 477 of SEQ ID NO:108, which is aconsensus sequence for HBV genotype B utilizing the design strategydiscussed above; (5) an optimized portion of HBV Core proteinrepresented by positions 478 to 629 of SEQ ID NO:108, which is aconsensus sequence for HBV genotype B utilizing the design strategydiscussed above; (6) an optimized portion of HBV X antigen representedby positions 630 to 689 of SEQ ID NO:108, which is a consensus sequencefor HBV genotype B utilizing the design strategy discussed above; and(7) an optional hexahistidine tag. A yeast-based immunotherapycomposition expressing this complete fusion protein is also referred toherein as GI-13011. The fusion protein and corresponding yeast-basedimmunotherapeutic can also be referred to herein as “SPEXv2-B” or“Spex-B”.

A third construct based on a consensus sequence for HBV genotype C wasdesigned as follows. Using SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99 andSEQ ID NO:100, which were designed to reduce the size of the fusionsegments (as compared to full-length), to maximize the inclusion ofknown T cell epitopes corresponding to those identified in Table 5(priority as discussed above), and to minimize artificial junctions, newfusion segments were created based on a consensus sequence for HBVgenotype C. The new surface antigen segment is represented by positions1-249 of SEQ ID NO:109. The new polymerase (RT) segment is representedby positions 250-477 of SEQ ID NO:109. The new Core segment isrepresented by positions 478-629 of SEQ ID NO:109. The new X antigensegment is represented by positions 630-689 of SEQ ID NO:109. Thisfusion protein is a single polypeptide with the following sequenceelements fused in frame from N- to C-terminus, represented by SEQ IDNO:109 (non-HBV sequences denoted as “optional” were not included in thebase sequence of SEQ ID NO:109, but were actually added to the fusionprotein described in this example): (1) an optional N-terminal peptidethat is a synthetic N-terminal peptide designed to impart resistance toproteasomal degradation and stabilize expression represented by SEQ IDNO:37; (2) an optional linker peptide of Thr-Ser; (3) an optimizedportion of an HBV large (L) surface antigen represented by positions 1to 249 of SEQ ID NO:109, which is a consensus sequence for HBV genotypeC utilizing the design strategy discussed above; (4) an optimizedportion of the reverse transcriptase (RT) domain of HBV polymeraserepresented by positions 250 to 477 of SEQ ID NO:109, which is aconsensus sequence for HBV genotype C utilizing the design strategydiscussed above; (5) an optimized portion of HBV Core proteinrepresented by positions 478 to 629 of SEQ ID NO:109, which is aconsensus sequence for HBV genotype C utilizing the design strategydiscussed above; (6) an optimized portion of HBV X antigen representedby positions 630 to 689 of SEQ ID NO:109, which is a consensus sequencefor HBV genotype C utilizing the design strategy discussed above; and(7) an optional hexahistidine tag. A yeast-based immunotherapycomposition expressing this complete fusion protein is also referred toherein as GI-13012. The fusion protein and corresponding yeast-basedimmunotherapeutic can also be referred to herein as “SPEXv2-C” or“Spex-C”.

A fourth construct based on a consensus sequence for HBV genotype D wasdesigned as follows. Using SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99 andSEQ ID NO:100, which were designed to reduce the size of the fusionsegments (as compared to full-length), to maximize the inclusion ofknown T cell epitopes corresponding to those identified in Table 5(priority as discussed above), and to minimize artificial junctions, newfusion segments were created based on a consensus sequence for HBVgenotype D. The new surface antigen segment is represented by positions1-249 of SEQ ID NO:110. The new polymerase (RT) segment is representedby positions 250-477 of SEQ ID NO:110. The new Core segment isrepresented by positions 478-629 of SEQ ID NO:110. The new X antigensegment is represented by positions 630-689 of SEQ ID NO:110. Thisfusion protein is a single polypeptide with the following sequenceelements fused in frame from N- to C-terminus, represented by SEQ IDNO:110 (non-HBV sequences denoted as “optional” were not included in thebase sequence of SEQ ID NO:110, but were actually added to the fusionprotein described in this example): (1) an optional N-terminal peptidethat is a synthetic N-terminal peptide designed to impart resistance toproteasomal degradation and stabilize expression represented by SEQ IDNO:37; (2) an optional linker peptide of Thr-Ser; (3) an optimizedportion of an HBV large (L) surface antigen represented by positions 1to 249 of SEQ ID NO: 110, which is a consensus sequence for HBV genotypeD utilizing the design strategy discussed above; (4) an optimizedportion of the reverse transcriptase (RT) domain of HBV polymeraserepresented by positions 250 to 477 of SEQ ID NO: 110, which is aconsensus sequence for HBV genotype D utilizing the design strategydiscussed above; (5) an optimized portion of HBV Core proteinrepresented by positions 478 to 629 of SEQ ID NO: 110, which is aconsensus sequence for HBV genotype D utilizing the design strategydiscussed above; (6) an optimized portion of HBV X antigen representedby positions 630 to 689 of SEQ ID NO: 110, which is a consensus sequencefor HBV genotype D utilizing the design strategy discussed above; and(7) an optional hexahistidine tag. A yeast-based immunotherapycomposition expressing this complete fusion protein is also referred toherein as GI-13013. A yeast-based immunotherapy composition expressing asimilar fusion protein (containing SEQ ID NO:110), except that theN-terminal peptide of SEQ ID NO:37 is substituted with the alpha factorsequence of SEQ ID NO:89, is referred to herein as GI-13014. The fusionprotein and corresponding yeast-based immunotherapeutic can also bereferred to herein as “SPEXv2-D”, “Spex-D”, or “M-SPEXv2-D” (forGI-13013) or “a-SPEXv2-D” for (GI-13014).

Additional HBV fusion proteins for use in a yeast-basedimmunotherapeutic were designed using the application of consensussequences for four HBV genotypes to demonstrate how alterations similarto those made in the fusion proteins described above (SEQ ID NOs:107-110) can be made in a different HBV fusion protein, such as thatdescribed by SEQ ID NO:34, which contains HBV Surface proteins and HBVCore proteins. To design these additional HBV antigens and correspondingyeast-based immunotherapy compositions, the fusion protein structuredescribed above for SEQ ID NO:34 (and therefore the subparts of thesefusion proteins (Surface antigen and Core) was used as a template. Asabove for the constructs described above, consensus sequences for eachof HBV genotype A, B, C and D were built from multiple sources of HBVsequences (e.g., Yu and Yuan et al, 2010, for S and Core), and sequencesin the template structure were replaced with consensus sequencescorresponding to the same positions, unless using the consensus sequencealtered one of the known acute self-limiting T cells epitopes or one ofthe known polymerase escape mutation sites, in which case, thesepositions followed the published sequence for these epitopes or mutationsites.

A first construct based on a consensus sequence for HBV genotype A wasdesigned as follows. Using SEQ ID NO:34 as a template, a new fusionprotein was created based on a consensus sequence for HBV genotype A,represented here by SEQ ID NO:112. This fusion protein is a singlepolypeptide with the following sequence elements fused in frame from N-to C-terminus, represented by SEQ ID NO:112 (non-HBV sequences denotedas “optional” are not included in the base sequence of SEQ ID NO:112,but were actually added to the fusion protein described in thisexample): (1) an optional N-terminal peptide that is a syntheticN-terminal peptide designed to impart resistance to proteasomaldegradation and stabilize expression represented by SEQ ID NO:37; (2) anoptional linker peptide of Thr-Ser; (3) a consensus sequence for HBVgenotype A large (L) surface antigen represented by positions 1 to 399of SEQ ID NO:112; 4) the amino acid sequence of a consensus sequence forHBV genotype A core antigen represented by positions 400 to 581 of SEQID NO:112; and (5) an optional hexahistidine tag. A nucleic acidsequence encoding the fusion protein comprising SEQ ID NO:112 (codonoptimized for yeast expression) is represented herein by SEQ ID NO:111.A yeast-based immunotherapy composition expressing this fusion proteinis also referred to herein as GI-13006. The fusion protein andcorresponding yeast-based immunotherapeutic can also be referred toherein as “Score-A”.

A second construct based on a consensus sequence for HBV genotype B wasdesigned as follows. Using SEQ ID NO:34 as a template, a new fusionprotein was created based on a consensus sequence for HBV genotype B,represented here by SEQ ID NO:114. This fusion protein is a singlepolypeptide with the following sequence elements fused in frame from N-to C-terminus, represented by SEQ ID NO:114 (non-HBV sequences denotedas “optional” are not included in the base sequence of SEQ ID NO:114,but were actually added to the fusion protein described in thisexample): (1) an optional N-terminal peptide that is a syntheticN-terminal peptide designed to impart resistance to proteasomaldegradation and stabilize expression represented by SEQ ID NO:37; (2) anoptional linker peptide of Thr-Ser; (3) a consensus sequence for HBVgenotype B large (L) surface antigen represented by positions 1 to 399of SEQ ID NO:114; 4) the amino acid sequence of a consensus sequence forHBV genotype B core antigen represented by positions 400 to 581 of SEQID NO:114; and (5) an optional hexahistidine tag. A nucleic acidsequence encoding the fusion protein comprising SEQ ID NO:114 (codonoptimized for yeast expression) is represented herein by SEQ ID NO:113.A yeast-based immunotherapy composition expressing this fusion proteinis also referred to herein as GI-13007. The fusion protein andcorresponding yeast-based immunotherapeutic can also be referred toherein as “Score-B”.

A third construct based on a consensus sequence for HBV genotype C wasdesigned as follows. Using SEQ ID NO:34 as a template, a new fusionprotein was created based on a consensus sequence for HBV genotype C,represented here by SEQ ID NO:116. This fusion protein is a singlepolypeptide with the following sequence elements fused in frame from N-to C-terminus, represented by SEQ ID NO:116 (non-HBV sequences denotedas “optional” are not included in the base sequence of SEQ ID NO:116,but were actually added to the fusion protein described in thisexample): (1) an optional N-terminal peptide that is a syntheticN-terminal peptide designed to impart resistance to proteasomaldegradation and stabilize expression represented by SEQ ID NO:37; (2) anoptional linker peptide of Thr-Ser; (3) a consensus sequence for HBVgenotype C large (L) surface antigen represented by positions 1 to 399of SEQ ID NO:116; 4) the amino acid sequence of a consensus sequence forHBV genotype C core antigen represented by positions 400 to 581 of SEQID NO:116; and (5) an optional hexahistidine tag. A nucleic acidsequence encoding the fusion protein comprising SEQ ID NO:116 (codonoptimized for yeast expression) is represented herein by SEQ ID NO:115.A yeast-based immunotherapy composition expressing this fusion proteinis also referred to herein as GI-13008. The fusion protein andcorresponding yeast-based immunotherapeutic can also be referred toherein as “Score-C”.

A fourth construct based on a consensus sequence for HBV genotype D wasdesigned as follows. Using SEQ ID NO:34 as a template, a new fusionprotein was created based on a consensus sequence for HBV genotype D,represented here by SEQ ID NO:118. This fusion protein is a singlepolypeptide with the following sequence elements fused in frame from N-to C-terminus, represented by SEQ ID NO:118 (non-HBV sequences denotedas “optional” are not included in the base sequence of SEQ ID NO:118,but were actually added to the fusion protein described in thisexample): (1) an optional N-terminal peptide that is a syntheticN-terminal peptide designed to impart resistance to proteasomaldegradation and stabilize expression represented by SEQ ID NO:37; (2) anoptional linker peptide of Thr-Ser; (3) a consensus sequence for HBVgenotype D large (L) surface antigen represented by positions 1 to 399of SEQ ID NO:118; 4) the amino acid sequence of a consensus sequence forHBV genotype D core antigen represented by positions 400 to 581 of SEQID NO:118; and (5) an optional hexahistidine tag. The amino acidsequence of the complete fusion protein comprising SEQ ID NO:118 and theN- and C-terminal peptides and linker peptide is represented herein bySEQ ID NO:151. A nucleic acid sequence encoding the fusion proteincomprising SEQ ID NO:118 or SEQ ID NO:151 (codon optimized for yeastexpression) is represented herein by SEQ ID NO:117. A yeast-basedimmunotherapy composition expressing this fusion protein is alsoreferred to herein as GI-13009. The fusion proteins and correspondingyeast-based immunotherapeutic can also be referred to herein as“Score-D”.

The yeast-based immunotherapy compositions of GI-13010 (comprising SEQID NO:107), GI-13011 (comprising SEQ ID NO:108), GI-13012 (comprisingSEQ ID NO:109), GI-13013 (comprising SEQ ID NO:110), GI-13006(comprising SEQ ID NO:112), GI-13007 (comprising SEQ ID NO:114),GI-13008 (comprising SEQ ID NO:116) and GI-13009 (comprising SEQ IDNO:118) were produced as described for other compositions above.Briefly, DNA encoding the fusion protein was codon optimized forexpression in yeast and then inserted behind the CUP1 promoter (pGI-100)in yeast 2 um expression vectors. The resulting plasmids were introducedinto Saccharomyces cerevisiae W303α yeast by Lithiumacetate/polyethylene glycol transfection. Yeast transformants of eachplasmid were isolated on solid minimal plates lacking uracil (UDM;uridine dropout medium). Colonies were re-streaked onto ULDM (uridineand leucine dropout medium) and allowed to grow for 3 days at 30° C.Liquid starter cultures lacking uridine and leucine (UL2; formulationprovided in Example 1) were inoculated from plates and starter cultureswere grown for 18 h at 30° C., 250 rpm. Primary cultures were used toinoculate final cultures of UL2 and growth continued until a density of2 YU/mL was reached. Cultures were induced with 0.5 mM copper sulfatefor 3 h and then cells were washed in PBS, heat-killed at 56° C. for 1h, and washed three times in PBS. Total protein content was measured bya TCA precipitation/nitrocellulose binding assay and HBV antigenexpression was measured by western blot using an anti-his tag monoclonalantibody.

The results are shown in FIG. 20. The lanes in the blot shown in FIG. 20contain protein from the following yeast-based immunotherapeutics: Lane1 (v1.0; Score)=GI-13002 (expressing SEQ ID NO:34); Lane 2 (v2.0;ScA)=GI-13006 (expressing SEQ ID NO:112); Lane 3 (v2.0; ScB)=GI-13007(expressing SEQ ID NO:114); Lane 4 (v2.0; ScC)=GI-13008 (expressing SEQID NO:116); Lane 5 (v2.0; ScD)=GI-13009 (expressing SEQ ID NO:118); Lane6 (v1.0; Sp)=GI-13005 (expressing SEQ ID NO:36); Lane 7 (v1.0;a-Sp)=GI-13004 (expressing SEQ ID NO:92); Lane 8 (v2.0; SpA)=GI-13010(expressing SEQ ID NO:107); Lane 9 (v2.0; SpB)=GI-13011 (expressing SEQID NO:108); Lane 10 (v2.0; SpC)=GI-13012 (expressing SEQ ID NO:109);Lane 11 (v2.0; SpD)=GI-13013 (expressing SEQ ID NO:110).

The results show that each of the HBV antigens comprising thecombination of surface antigen and core (“Score” antigens), i.e.,GI-13002 (Score), GI-13006 (ScA; Score-A), GI-13007 (ScB; Score-B),GI-13008 (ScC; Score-C), and GI-13009 (ScD; Score-D) expressed robustlyin yeast. Typical Score v2.0 expression levels in these and similarexperiments were in the range of approximately 90 to 140 pmol/YU (i.e.,5940 ng/YU to 9240 ng/YU). Expression levels of the HBV antigenscomprising all four HBV proteins (surface, polymerase, core and X, or“Spex”) was variable. Specifically, expression of the antigens fromGI-13010 (SpA; Spex-A), GI-13011 (SpB; Spex-B), GI-13012 (SpC; Spex-D)and GI-13013 (SpD; Spex-D) was substantially lower than expression ofthe “Score” antigens, as well as the antigens from GI-13005 (Sp; Spex)and GI-13004 (a-Sp; a-Spex). Expression of the antigen in GI-13012 (SpC;Spex-C) was barely detectable. Taken together, these results indicatethat as a group, HBV antigens comprising surface antigen and coreexpress very well in yeast, whereas HBV antigens comprising all ofsurface antigen, polymerase, core and X have variable expression inyeast, and generally express less well than the “Score” antigens.

Example 8

The following example describes the production of additional yeast-basedHBV immunotherapeutic compositions that utilize consensus sequences forHBV genotypes, and additionally demonstrate the use of alternateconfigurations/arrangements of HBV protein segments within a fusionprotein in order to modify or improve the expression of an HBV antigenin yeast and/or improve or modify the immunogenicity or other functionalattribute of the HBV antigen.

In this example, new fusion proteins were designed that append X antigenand/or polymerase antigens to the N- or C-terminus of the combination ofsurface antigen fused to core. These constructs were designed in partbased on the rationale that because the fusion proteins arranged in theconfiguration generally referred to herein as “Score” (e.g., SEQ IDNO:34, SEQ ID NO:112, SEQ ID NO:114, SEQ ID NO:116 and SEQ ID NO:118)express very well in yeast, it may be advantageous to utilize this baseconfiguration (i.e., surface antigen fused to core protein) to produceHBV antigens comprising additional HBV protein components. Suchstrategies may improve expression of multi-protein antigens and/orimprove or modify the functionality of such antigens in the context ofimmunotherapy. For example, without being bound by theory, the inventorsproposed that the expression of an HBV antigen using three or all fourHBV proteins could be improved by constructing the fusion protein usingsurface-core (in order) as a base, and then appending the other antigensto this construct.

Accordingly, to exemplify this embodiment of the invention, eight newfusion proteins were designed and constructed, and yeast-basedimmunotherapy products expressing these proteins were produced. In eachcase, the fusion protein used a surface-core fusion protein as a basethat was derived from segments of the fusion protein represented by SEQID NO:118, which is a surface-core fusion protein described in Example 7utilizing a consensus sequence for HBV genotype D and optimized tomaximize the use of conserved immunological epitopes. All possiblearrangements of a polymerase segment and/or an X antigen segment wereappended to this base configuration, utilizing segments derived from thefusion protein represented by SEQ ID NO:110, which is a multi-proteinHBV fusion protein described in Example 7 that was constructed to reducethe size of the protein segments, maximize the use of conservedimmunological epitopes, and utilize a consensus sequence for HBVgenotype D. While these eight resulting antigens are based on aconsensus sequence for HBV genotype D, it would be straightforward toproduce a fusion protein having a similar overall structure using thecorresponding fusion segments from the fusion proteins represented bySEQ ID NO:107 and/or SEQ ID NO:112 (genotype A), SEQ ID NO:108 and/orSEQ ID NO:114 (genotype B), SEQ ID NO:109 and/or SEQ ID NO:116 (genotypeC), or using the corresponding sequences from a different HBV genotype,sub-genotype, consensus sequence or strain.

To produce the first composition, yeast (e.g., Saccharomyces cerevisiae)were engineered to express a new HBV fusion protein, schematicallyillustrated in FIG. 8, under the control of the copper-induciblepromoter, CUP1. The resulting yeast-HBV immunotherapy composition can bereferred to herein as GI-13015. This fusion protein, also referred toherein as “Score-Pol” and represented by SEQ ID NO:120, comprises, inorder, surface antigen, core protein, and polymerase sequences, as asingle polypeptide with the following sequence elements fused in framefrom N- to C-terminus (non-HBV sequences denoted as “optional” were notincluded in the base sequence of SEQ ID NO:120, but were actually addedto the fusion protein described in this example): (1) an optionalN-terminal peptide that is a synthetic N-terminal peptide designed toimpart resistance to proteasomal degradation and stabilize expressionrepresented by SEQ ID NO:37; (2) an optional linker peptide of Thr-Ser;(3) the amino acid sequence of a near full-length (minus position 1)consensus sequence for HBV genotype D large (L) surface antigenrepresented by positions 1 to 399 of SEQ ID NO:120 (corresponding topositions 1 to 399 of SEQ ID NO:118); (4) the amino acid sequence of aconsensus sequence for HBV genotype D core antigen represented bypositions 400 to 581 of SEQ ID NO:120 (corresponding to positions 400 to581 of SEQ ID NO:118); (5) an optimized portion of the reversetranscriptase (RT) domain of HBV polymerase using a consensus sequencefor HBV genotype D, represented by positions 582 to 809 of SEQ ID NO:120(corresponding to positions to 250 to 477 of SEQ ID NO:110); and (6) anoptional hexahistidine tag. SEQ ID NO:120 contains multiple T cellepitopes (human and murine), which can be found in Table 5. A nucleicacid sequence encoding the fusion protein of SEQ ID NO:120(codon-optimized for expression in yeast) is represented herein by SEQID NO:119.

To produce the second composition, yeast (e.g., Saccharomycescerevisiae) were engineered to express a new HBV fusion protein,schematically illustrated in FIG. 9, under the control of thecopper-inducible promoter, CUP1. The resulting yeast-HBV immunotherapycomposition can be referred to herein as GI-13016. This fusion protein,also referred to herein as “Score-X” and represented by SEQ ID NO:122,comprises, in order, surface antigen, core, and X antigen sequences, asa single polypeptide with the following sequence elements fused in framefrom N- to C-terminus (non-HBV sequences denoted as “optional” were notincluded in the base sequence of SEQ ID NO:122, but were actually addedto the fusion protein described in this example): (1) an optionalN-terminal peptide that is a synthetic N-terminal peptide designed toimpart resistance to proteasomal degradation and stabilize expressionrepresented by SEQ ID NO:37; (2) an optional linker peptide of Thr-Ser;(3) the amino acid sequence of a near full-length (minus position 1)consensus sequence for HBV genotype D large (L) surface antigenrepresented by positions 1 to 399 of SEQ ID NO:122 (corresponding topositions 1 to 399 of SEQ ID NO:118); 4) the amino acid sequence of aconsensus sequence for HBV genotype D core antigen represented bypositions 400 to 581 of SEQ ID NO:122 (corresponding to positions 400 to581 of SEQ ID NO:118); (5) an optimized portion of HBV X antigen using aconsensus sequence for HBV genotype D, represented by positions 582 to641 of SEQ ID NO:122 (corresponding to positions 630 to 689 of SEQ IDNO:110); and (6) an optional hexahistidine tag. SEQ ID NO:122 containsmultiple T cell epitopes (human and murine), which can be found in Table5. A nucleic acid sequence encoding the fusion protein comprising SEQ IDNO:122 (codon-optimized for expression in yeast) is represented hereinby SEQ ID NO:121.

To produce the third composition, yeast (e.g., Saccharomyces cerevisiae)were engineered to express a new HBV fusion protein, schematicallyillustrated in FIG. 10, under the control of the copper-induciblepromoter, CUP1. The resulting yeast-HBV immunotherapy composition can bereferred to herein as GI-13017. This fusion protein, also referred toherein as “Score-Pol-X” and represented by SEQ ID NO:124 comprises, inorder, surface antigen, core, polymerase and X antigen sequences, as asingle polypeptide with the following sequence elements fused in framefrom N- to C-terminus (non-HBV sequences denoted as “optional” were notincluded in the base sequence of SEQ ID NO:124, but were actually addedto the fusion protein described in this example): (1) an optionalN-terminal peptide that is a synthetic N-terminal peptide designed toimpart resistance to proteasomal degradation and stabilize expressionrepresented by SEQ ID NO:37; (2) an optional linker peptide of Thr-Ser;(3) the amino acid sequence of a near full-length (minus position 1)consensus sequence for HBV genotype D large (L) surface antigenrepresented by positions 1 to 399 of SEQ ID NO:124 (corresponding topositions 1 to 399 of SEQ ID NO:118); 4) the amino acid sequence of aconsensus sequence for HBV genotype D core antigen represented bypositions 400 to 581 of SEQ ID NO:124 (corresponding to positions 400 to581 of SEQ ID NO:118); (5) an optimized portion of the reversetranscriptase (RT) domain of HBV polymerase using a consensus sequencefor HBV genotype D, represented by positions 582 to 809 of SEQ ID NO:124(corresponding to positions to 250 to 477 of SEQ ID NO:110); (6) anoptimized portion of HBV X antigen using a consensus sequence for HBVgenotype D, represented by positions 810 to 869 of SEQ ID NO:124(corresponding to positions 630 to 689 of SEQ ID NO:110); and (7) anoptional hexahistidine tag. SEQ ID NO:124 contains multiple T cellepitopes (human and murine), which can be found in Table 5. A nucleicacid sequence encoding the fusion protein comprising SEQ ID NO:124(codon-optimized for expression in yeast) is represented herein by SEQID NO:123.

To produce the fourth composition, yeast (e.g., Saccharomycescerevisiae) were engineered to express a new HBV fusion protein,schematically illustrated in FIG. 11, under the control of thecopper-inducible promoter, CUP1. The resulting yeast-HBV immunotherapycomposition can be referred to herein as GI-13018. This fusion protein,also referred to herein as “Score-X-Pol” and represented by SEQ IDNO:126 comprises, in order, surface antigen, core, X antigen, andpolymerase sequences, as a single polypeptide with the followingsequence elements fused in frame from N- to C-terminus (non-HBVsequences denoted as “optional” were not included in the base sequenceof SEQ ID NO:126, but were actually added to the fusion proteindescribed in this example): (1) an optional N-terminal peptide that is asynthetic N-terminal peptide designed to impart resistance toproteasomal degradation and stabilize expression represented by SEQ IDNO:37; (2) an optional linker peptide of Thr-Ser; (3) the amino acidsequence of a near full-length (minus position 1) consensus sequence forHBV genotype D large (L) surface antigen represented by positions 1 to399 of SEQ ID NO:126 (corresponding to positions 1 to 399 of SEQ IDNO:118); 4) the amino acid sequence of a consensus sequence for HBVgenotype D core antigen represented by positions 400 to 581 of SEQ IDNO:126 (corresponding to positions 400 to 581 of SEQ ID NO:118); (5) anoptimized portion of HBV X antigen using a consensus sequence for HBVgenotype D, represented by positions 582 to 641 of SEQ ID NO:126(corresponding to positions 630 to 689 of SEQ ID NO:110); (5) anoptimized portion of the reverse transcriptase (RT) domain of HBVpolymerase using a consensus sequence for HBV genotype D, represented bypositions 642 to 869 of SEQ ID NO:126 (corresponding to positions to 250to 477 of SEQ ID NO:110); and (7) an optional hexahistidine tag. SEQ IDNO:126 contains multiple T cell epitopes (human and murine), which canbe found in Table 5. A nucleic acid sequence encoding the fusion proteincomprising SEQ ID NO:126 (codon-optimized for expression in yeast) isrepresented herein by SEQ ID NO:125.

To produce the fifth composition, yeast (e.g., Saccharomyces cerevisiae)were engineered to express a new HBV fusion protein, schematicallyillustrated in FIG. 12, under the control of the copper-induciblepromoter, CUP1. The resulting yeast-HBV immunotherapy composition can bereferred to herein as GI-13019. This fusion protein, also referred toherein as “Pol-Score” and represented by SEQ ID NO:128 comprises, inorder, polymerase, surface antigen, and core sequences, as a singlepolypeptide with the following sequence elements fused in frame from N-to C-terminus (non-HBV sequences denoted as “optional” were not includedin the base sequence of SEQ ID NO:128, with the exception of the Leu-Glulinker between the polymerase segment and the surface antigen segment inthe construct exemplified here, but were actually added to the fusionprotein described in this example): (1) an optional N-terminal peptidethat is a synthetic N-terminal peptide designed to impart resistance toproteasomal degradation and stabilize expression represented by SEQ IDNO:37; (2) an optional linker peptide of Thr-Ser; (3) an optimizedportion of the reverse transcriptase (RT) domain of HBV polymerase usinga consensus sequence for HBV genotype D, represented by positions 1 to228 of SEQ ID NO:120 (corresponding to positions to 250 to 477 of SEQ IDNO:110); (4) a linker peptide (optional) of Leu-Glu, represented bypositions 229 to 230 of SEQ ID NO:128; (5) the amino acid sequence of anear full-length (minus position 1) consensus sequence for HBV genotypeD large (L) surface antigen represented by positions 231 to 629 of SEQID NO:128 (corresponding to positions 1 to 399 of SEQ ID NO:118); (6)the amino acid sequence of a consensus sequence for HBV genotype D coreantigen represented by positions 630 to 811 of SEQ ID NO:128(corresponding to positions 400 to 581 of SEQ ID NO:118); and (7) anoptional hexahistidine tag. SEQ ID NO:128 contains multiple T cellepitopes (human and murine), which can be found in Table 5. A nucleicacid sequence encoding the fusion protein comprising SEQ ID NO:128(codon-optimized for expression in yeast) is represented herein by SEQID NO:127.

To produce the sixth composition, yeast (e.g., Saccharomyces cerevisiae)were engineered to express a new HBV fusion protein, schematicallyillustrated in FIG. 13, under the control of the copper-induciblepromoter, CUP1. The resulting yeast-HBV immunotherapy composition can bereferred to herein as GI-13020. This fusion protein, also referred toherein as “X-Score” and represented by SEQ ID NO:130 comprises, inorder, X antigen, surface antigen, and core sequences, as a singlepolypeptide with the following sequence elements fused in frame from N-to C-terminus, (non-HBV sequences denoted as “optional” were notincluded in the base sequence of SEQ ID NO:130, with the exception ofthe Leu-Glu linker between the X segment and the surface antigen segmentin the construct exemplified here, but were actually added to the fusionprotein described in this example): (1) an optional N-terminal peptidethat is a synthetic N-terminal peptide designed to impart resistance toproteasomal degradation and stabilize expression represented by SEQ IDNO:37; (2) an optional linker peptide of Thr-Ser; (3) an optimizedportion of HBV X antigen using a consensus sequence for HBV genotype D,represented by positions 1 to 60 of SEQ ID NO:130 (corresponding topositions 630 to 689 of SEQ ID NO:110); (4) a linker peptide (optional)of Leu-Glu, represented by positions 61 to 62 of SEQ ID NO:130; (5) theamino acid sequence of a near full-length (minus position 1) consensussequence for HBV genotype D large (L) surface antigen represented bypositions 63 to 461 of SEQ ID NO:130 (corresponding to positions 1 to399 of SEQ ID NO:118); (6) the amino acid sequence of a consensussequence for HBV genotype D core antigen represented by positions 462 to643 of SEQ ID NO:130 (corresponding to positions 400 to 581 of SEQ IDNO:118); and (7) an optional hexahistidine tag. SEQ ID NO:130 containsmultiple T cell epitopes (human and murine), which can be found in Table5. The amino acid sequence of the complete fusion protein comprising SEQID NO:130 and the N- and C-terminal peptides and linkers is representedherein by SEQ ID NO:150. A nucleic acid sequence encoding the fusionprotein comprising SEQ ID NO:130 or SEQ ID NO:150 (codon-optimized forexpression in yeast) is represented herein by SEQ ID NO:129.

To produce the seventh composition, yeast (e.g., Saccharomycescerevisiae) were engineered to express a new HBV fusion protein,schematically illustrated in FIG. 14, under the control of thecopper-inducible promoter, CUP1. The resulting yeast-HBV immunotherapycomposition can be referred to herein as GI-13021. This fusion protein,also referred to herein as “Pol-X-Score” and represented by SEQ IDNO:132 comprises, in order, polymerase, X antigen, surface antigen, andcore, as a single polypeptide with the following sequence elements fusedin frame from N- to C-terminus (non-HBV sequences denoted as “optional”were not included in the base sequence of SEQ ID NO:132, but wereactually added to the fusion protein described in this example): (1) anoptional N-terminal peptide that is a synthetic N-terminal peptidedesigned to impart resistance to proteasomal degradation and stabilizeexpression represented by SEQ ID NO:37; (2) an optional linker peptideof Thr-Ser; (3) an optimized portion of the reverse transcriptase (RT)domain of HBV polymerase using a consensus sequence for HBV genotype D,represented by positions 1 to 228 of SEQ ID NO:132 (corresponding topositions to 250 to 477 of SEQ ID NO:110); (4) an optimized portion ofHBV X antigen using a consensus sequence for HBV genotype D, representedby positions 229 to 288 of SEQ ID NO:132 (corresponding to positions 630to 689 of SEQ ID NO:110); (5) the amino acid sequence of a nearfull-length (minus position 1) consensus sequence for HBV genotype Dlarge (L) surface antigen represented by positions 289 to 687 of SEQ IDNO:132 (corresponding to positions 1 to 399 of SEQ ID NO:118); (6) theamino acid sequence of a consensus sequence for HBV genotype D coreantigen represented by positions 688 to 869 of SEQ ID NO:132(corresponding to positions 400 to 581 of SEQ ID NO:118); and (7) anoptional hexahistidine tag. SEQ ID NO:132 contains multiple T cellepitopes (human and murine), which can be found in Table 5. A nucleicacid sequence encoding the fusion protein comprising SEQ ID NO:132(codon-optimized for expression in yeast) is represented herein by SEQID NO:131.

To produce the eighth composition, yeast (e.g., Saccharomycescerevisiae) were engineered to express a new HBV fusion protein,schematically illustrated in FIG. 15, under the control of thecopper-inducible promoter, CUP1. The resulting yeast-HBV immunotherapycomposition can be referred to herein as GI-13022. This fusion protein,also referred to herein as “X-Pol-Score” and represented by SEQ IDNO:134 comprises, in order, X antigen, polymerase, surface antigen, andcore protein, as a single polypeptide with the following sequenceelements fused in frame from N- to C-terminus (non-HBV sequences denotedas “optional” were not included in the base sequence of SEQ ID NO:134,but were actually added to the fusion protein described in thisexample): (1) an optional N-terminal peptide that is a syntheticN-terminal peptide designed to impart resistance to proteasomaldegradation and stabilize expression represented by SEQ ID NO:37; (2) anoptional linker peptide of Thr-Ser; (3) an optimized portion of HBV Xantigen using a consensus sequence for HBV genotype D, represented bypositions 1 to 60 of SEQ ID NO:134 (corresponding to positions 630 to689 of SEQ ID NO:110); (4) an optimized portion of the reversetranscriptase (RT) domain of HBV polymerase using a consensus sequencefor HBV genotype D, represented by positions 61 to 288 of SEQ ID NO:134(corresponding to positions to 250 to 477 of SEQ ID NO:110); (5) theamino acid sequence of a near full-length (minus position 1) consensussequence for HBV genotype D large (L) surface antigen represented bypositions 289 to 687 of SEQ ID NO:134 (corresponding to positions 1 to399 of SEQ ID NO:118); (6) the amino acid sequence of a consensussequence for HBV genotype D core antigen represented by positions 688 to869 of SEQ ID NO:134 (corresponding to positions 400 to 581 of SEQ IDNO:118); and (7) an optional hexahistidine tag. SEQ ID NO:134 containsmultiple T cell epitopes (human and murine), which can be found in Table5. A nucleic acid sequence encoding the fusion protein comprising SEQ IDNO:134 (codon-optimized for expression in yeast) is represented hereinby SEQ ID NO:133.

To produce each of the yeast-based immunotherapy compositions describedabove, yeast transformants of each plasmid were isolated on solidminimal plates lacking uracil (UDM; uridine dropout medium). Colonieswere re-streaked onto ULDM and UDM plates and allowed to grow for 3 daysat 30° C. Liquid starter cultures lacking uridine and leucine (UL2) orlacking uridine (U2) were inoculated from plates and starter cultureswere grown for 18 h at 30° C., 250 rpm. Primary cultures were used toinoculate intermediate cultures of U2 or UL2 and growth was continueduntil a density of approximately 2 YU/mL was reached. Intermediatecultures were used to inoculate final cultures to a density of 0.05YU/mL and these were incubated until the cell density reached 1-3 YU/mL.Final cultures were then induced with 0.5 mM copper sulfate for 3 h andcells were washed in PBS, heat killed at 56° C. for 1 h, and washedthree times in PBS. Total protein content was measured with a TCAprecipitation/nitrocellulose binding assay and HBV antigen expressionwas measured by Western blot using an anti-his tag monoclonal antibody.Lysates from two yeast immunotherapeutic compositions described inExample 7 as GI-13008 (SEQ ID NO:116; “Score-C”) or GI-13009 (SEQ IDNO:118; “Score-D”) were used as a basis of comparison to a yeastexpressing the base surface-core antigen product.

FIG. 21 is a blot showing the expression of all eight constructs inyeast cultured in UL2 medium (1 μg of protein loaded) as compared toexpression of the construct in the yeast immunotherapeutic described inExample 7 as GI-13009 (SEQ ID NO:118; “Score-D”). Referring to FIG. 21,lanes 1 and 2 contain molecular weight markers, and lanes 4-6 containrecombinant hexahistidine tagged NS3 protein that was processed on thesame blot in order to quantify antigen by interpolation from a standardcurve generated from these lanes. Lanes 7-14 contain lysates from eachyeast-based immunotherapeutic denoted by number (e.g., GI-13015) grownin UL2 medium, and lane 15 contains the lysate from the GI-13009comparison. Additional western blots from yeast cultured in U2 medium,as well as additional blots evaluating different amounts of proteinloading on the gel are not shown here, but overall, the resultsindicated that all eight antigens were expressed to detectable levels inat least one growth medium.

The overall expression results are summarized in FIG. 22 as a bar graphfor those cultures that had detectable expression of target antigen inU2 or UL2 medium as compared to expression of antigens in GI-13008(Score-C) and GI-13009 (Score-D). Referring to FIG. 22, the HBV antigensare denoted below each bar using the reference to antigen arrangement inthe fusion protein as described for each construct above, along with themedium used to culture the corresponding yeast that expressed theantigen (i.e., “Pol-Score-U2” refers to the HBV antigen that is apolymerase-surface-core fusion protein, represented by SEQ ID NO:128 andexpressed by GI-13019 in U2 medium). The results indicated thatexpression of the antigen denoted “X-Score” (expressed by GI-13020; SEQID NO:130) was particularly robust, at ˜122 pmol/YU, which wasapproximately 79-80% of the expression level obtained for Score-C(GI-13008) or Score-D (GI-13009) on a molar basis (either medium).Expression of the antigens expressed by GI-13015 (Score-Pol; SEQ IDNO:120), GI-13016 (Score-X; SEQ ID NO:122), GI-13017 (Score-Pol-X; SEQID NO:124) and GI-13018 (Score-X-Pol; SEQ ID NO:126) in U2 medium wasbelow the level of quantification in this experiment, although each ofthese antigens were expressed when the same yeast-basedimmunotherapeutic was grown in UL2 medium (see FIG. 22). In general,antigen configurations containing the polymerase reverse transcriptase(RT) domain accumulated to lower levels than those containing onlyS-core with or without the addition of X antigen. Taking the data shownin this and prior Examples as a whole, the antigen configurations ofsurface-core (“Score” or “SCORE”; all similar constructs) andX-surface-core (“X-Score” or “X-SCORE”; GI-13020) were the highestexpressing antigen configurations among all yeast-based HBVimmunotherapeutics tested.

Example 9

The following example describes preclinical experiments in mice todemonstrate the safety, immunogenicity, and in vivo efficacy ofyeast-based HBV immunotherapy compositions of the invention.

To evaluate the yeast-based HBV immunotherapy compositions inpreclinical studies, a variety of in vitro and in vivo assays thatdetect induction of antigen-specific lymphocytes by yeast-based HBVimmunotherapy compositions of the invention were employed, includinglymphocyte proliferation, cell-mediated cytotoxicity, cytokinesecretion, and protection from tumor challenge (e.g., killing of tumorsengineered to express HBV proteins in vivo).

To support these studies, yeast-based HBV immunotherapy compositionsdescribed in Examples 1 and 2 were used initially, with additionalstudies performed using yeast-based HBV immunotherapy compositionsdescribed in 7 and 8 or elsewhere herein. However, these studies can bereadily applied to any yeast-based HBV immunotherapy composition of theinvention, and the results provided herein can be extrapolated to otherHBV compositions comprising the same antigen base or similar antigenconstructs. The results of these initial experiments are describedbelow.

As a general protocol that can be adapted for any yeast-based HBVimmunotherapy composition, mice (e.g., female BALB/c and/or C57BL/6mice) are injected with a suitable amount of a yeast-based HBVimmunotherapy composition, e.g., 4-5 YU (administered subcutaneously in2-2.5 YU injections at 2 different injection sites). Optionally, aninjection of anti-CD40 antibody is administered the day following theyeast compositions. Mice are immunized weekly or biweekly, for 1, 2, or3 doses, and a final booster dose is optionally administered 3-4 weeksafter the last weekly or biweekly dose. Mice are sacrificed 7-9 daysafter the final injection. Spleen cell suspensions, and/or lymph nodesuspensions, pooled from each group, are prepared and subjected to invitro stimulation (IVS) conditions utilizing HBV-specific stimuli in theform of HBV peptides and/or HBV antigens, which may include yeastexpressing HBV antigens. Control cultures are stimulated with non-HBVpeptides, which can include an ovalbumin peptide, or a non-relevantviral peptide (e.g., a peptide from HIV). Standard assays are employedto evaluate immune responses induced by administration of yeast-basedHBV immunotherapy compositions and include lymphocyte proliferation asassessed by ³H-thymidine incorporation, cell-mediated cytotoxicityassays (CTL assays) employing ⁵¹Cr-labeled target cells (or othertargets labeled for overnight CTL), quantification of cytokine secretionby cytokine assay or ELISPOT (e.g., IFN-γ, IL-12, TNF-α, IL-6, and/orIL-2, etc.), and protection from tumor challenge (e.g., in vivochallenge with tumor cells recombinantly engineered to express HBVantigens).

Yeast-based HBV immunotherapy compositions are expected to beimmunogenic as demonstrated by their ability to elicit HBVantigen-specific T cell responses as measured by the assays describedabove.

In initial experiments, two of the yeast-based HBV immunotherapyproducts described in Examples 1 and 2 were tested in lymphocyteproliferation assays (LPA) to determine whether immunization with theseproducts elicits antigen-specific CD4⁺ T cell proliferation. Morespecifically, the yeast-based immunotherapy product (GI-13002)expressing a fusion protein represented by SEQ ID NO:34 under thecontrol of the CUP1 promoter, also known as “SCORE” and morespecifically described in Example 1 above, and the yeast-basedimmunotherapy product (GI-13004) expressing a fusion protein representedby SEQ ID NO:92 under the control of the CUP1 promoter and also known as“a-SPEX” and more specifically described in Example 2, were each used toimmunize mice and evaluate CD4⁺ T cells specific for the surface and/orCore antigens that are targeted in both products using lymphocyteproliferation assays (LPAs).

Female BALB/c mice were immunized three times weekly with 5 YU of“SCORE” or a-SPEX subcutaneously at 2 different sites on the mouse (2.5YU/flank). Control mice were vaccinated with empty vector yeast (denoted“YVEC”) or nothing (denoted “Naïve”). One week after the thirdimmunization, mice were humanely sacrificed and spleens and periaortaland inguinal draining lymph nodes (LNs) were removed and processed tosingle cell suspensions. LN cells from the two types of nodes werepooled and stimulated in vitro (IVS) with a mixture of recombinant coreand surface antigen (“S/Core mix”) or a class II restricted mimetopepeptide (GYHGSSLY, SEQ ID NO:103, denoted “Class II SAg mimetopepeptide”), previously reported to elicit proliferation of T cells fromSAg-immunized BALB/c mice (Rajadhyaksha et al (1995). PNAS 92:1575-1579).

Spleen cells were subjected to CD4⁺ T cell enrichment by MagneticActivated Cell Sorting (MACS) and incubated with the same antigens asdescribed for LN. After 4 days incubation, IVS cultures were pulsed withtritiated (³H) thymidine for 18 h, and cellular DNA was harvested onglass fiber microfilters. The level of incorporated ³H-thymidine wasmeasured by scintillation counting. Replicate LN cultures fromSCORE-immunized mice were assayed in parallel. Interferon gamma (IFN-γ)production by ELISpot was used as an additional means to assess T cellactivation.

As shown in FIG. 23, FIG. 24 and FIG. 26, CD4⁺ T cells from SCORE- ora-SPEX-immunized mice proliferated in response to the recombinant S- andCore antigen mixture. Splenic T cells from SCORE-immunized mice (FIG.23) showed >5 fold higher level of proliferation than T cells fromYVEC-immunized (empty vector control) or Naive mice, indicating that theeffect is specific for the Surface-Core fusion protein (i.e.,antigen-specific T cell response). T cells from SCORE-immunized miceincubated with the HBV mimetope peptide also proliferated to higherlevels than peptide-pulsed YVEC or Naive controls, providing furtherevidence of the antigen-specificity of the yeast-based immunotherapeuticproduct response. These effects are also dependent upon the amount ofantigen added to IVS, with optimal activity occurring at 3 mg/ml(recombinant antigen) or 30 mg/mL (peptide).

As shown in FIG. 24, LN cells from SCORE-immunized mice alsoproliferated in response to IVS with these same antigens, although thedifference in proliferation between SCORE vs. Naive or YVEC-immunizedanimals was smaller than for isolated splenic CD4⁺ T cells.

The ELISpot data (FIG. 25) indicate that LN preparations fromSCORE-immunized mice re-stimulated with S+C mix contain >10-fold moreIFN-γ secreting cells than LNs from Naive animals. IVS with HBV peptide(SEQ ID NO:103) also elicited an IFN-γ response. Specifically, the SCORELN preps contained >3.5-fold more IFN-γ-producing cells than Naive LNpreps (FIG. 25). These data collectively show that SCORE (yeast-basedimmunotherapy expressing the fusion protein comprising surface antigenand core) elicits HBV antigen-specific T cell responses in both spleenand LN, and that these responses can be amplified by IVS with purifiedantigens in a dose-dependent fashion.

Similar analyses with a-SPEX (FIG. 26) showed that this yeast-based HBVimmunotherapeutic product also elicits T cell proliferative responses.a-SPEX elicited about a 30% increase as compared to YVEC in IVSperformed with the recombinant antigen mixture. Overall, the responsesobserved with a-SPEX were lower than those observed with SCORE. Thedifference in magnitude of the response may reflect the fact thatantigen expression in a-SPEX is less than half that of SCORE on a molarbasis. Alternatively, without being bound by theory, these results mayindicate that the configuration of the antigens expressed by the yeastinfluence expression level, processing efficiency through theendosome/proteasome, or other parameters of the immune response. Theproliferation of T cells from a-SPEX mice using the 100 mg/mL peptidewas at least 2-fold greater than the proliferation in YVEC vaccinatedmice (FIG. 26, right three columns).

Example 10

The following example describes the immunological evaluation of twoyeast-based HBV immunotherapeutics of the invention using cytokineprofiles.

One way to characterize the cellular immune response elicited as aresult of immunization with yeast-based HBV immunotherapeutics of theinvention is to evaluate the cytokine profiles produced upon ex vivostimulation of spleen preparations from the immunized animals.

In these experiments, female C57Bl/6 mice were immunized with GI-13002(“SCORE”, a yeast-based immunotherapeutic expressing the HBVsurface-core fusion protein represented by SEQ ID NO:34, Example 1) andGI-13005 (“M-SPEX”, a yeast-based immunotherapeutic expressing the HBVsurface-pol-core-X fusion protein represented by SEQ ID NO:36 under thecontrol of the CUP1 promoter, Example 2), YVEC (empty vector controlyeast), or nothing (Naïve) as follows: 2 YU of yeast-basedimmunotherapeutic or control yeast were injected subcutaneously at 2different sites on the animal on days 0, 7, & 28. Anti-CD40 antibody wasadministered by intraperitoneal (IP) injection on day 1 to provideadditional activation of dendritic cells (DCs) beyond the level ofactivation provided by yeast-based therapeutic. The anti-CD40 antibodytreatment is optional, but the use of the antibody can boost the levelof antigen-specific CD8⁺ T cells when attempting to detect these cellsby direct pentamer staining (such data not shown in this experiment).Nine days after the last immunization, spleens were removed andprocessed into single cell suspensions. The cells were put into in vitrostimulation (IVS) cultures for 48 h with a mixture of 2 HBV peptidespools (denoted “P” in FIG. 27 and FIG. 28 and “HBVP” in FIGS. 29A and29B), or with mitomycin C-treated naive syngeneic splenocytes pulsedwith the 2 peptides (denoted “PPS” in FIG. 27 and FIG. 28 and “HPPS” inFIGS. 29A and 29B). The peptides are H-2K^(b)-restricted and havefollowing sequences: ILSPFLPLL (SEQ ID NO:65, see Table 5) and MGLKFRQL(SEQ ID NO:104). The cultures were subjected to replicate Luminexanalysis of IL1β, IL-12, and IFN-γ.

These cytokines were evaluated because they are associated with thetypes of immune responses that are believed to be associated with aproductive or effective immune response against HBV. IL-1β is apro-inflammatory cytokine produced by antigen presenting cells, and is acytokine known to be induced by immunization with yeast-basedimmunotherapy compositions. IL-12 is also produced by antigen presentingcells and promotes CD8⁺ cytotoxic T lymphocyte (CTL) activity. IFN-γ isproduced by CD8⁺ cytotoxic T lymphocytes in the development of theadaptive immune response and also promoted Th1 CD4⁺ T celldifferentiation.

The results, shown in FIG. 27 (IL-1β), FIG. 28 (IL-12), FIG. 29A (IFN-γ;SCORE-immunized), and FIG. 29B (IFN-γ; M-SPEX-immunized) show that allthree cytokines are produced by splenocytes from Score-immunized mice(denoted “Sc” in FIG. 27, FIG. 28 and FIG. 29A) in response to directIVS with peptide pool alone, and that the response is greater forSCORE-immunized than for YVEC (denoted “Y” in FIG. 27, FIG. 28 and FIGS.29A and 29B) or Naive (denoted “N” in FIG. 27, FIG. 28 and FIGS. 29A and29B) mice, demonstrating that immunization with SCORE elicits anantigen-specific immune response resulting in production of these threecytokines. IVS with peptide-pulsed syngeneic splenocytes also elicitedan antigen specific response although of lower magnitude. Splenocytesfrom M-SPEX-vaccinated mice (denoted “Sp” in FIG. 27, FIG. 28 and FIG.29B) produced an overall lower level of the cytokines than those fromSCORE-vaccinated mice. Nevertheless, the amount of IL12p70 produced inresponse to M-SPEX is higher than the amount produced by YVEC or Naïve,indicating an antigen-specific immune response induced by thisyeast-based immunotherapeutic composition. It is expected that a-SPEX(GI-13004; Example 2), which expressed higher levels of antigen andinduced a CD4⁺ proliferative response in the assays described in Example9, will elicit higher levels of cytokine production.

Additional cytokine assays were performed using female BALB/c miceimmunized with one of the same two yeast-based immunotherapeuticproducts. In these experiments, female BALB/c mice were immunized withSCORE (GI-13002; denoted “Sc” in FIGS. 30A-30D), M-SPEX (GI-13005;denoted “Sp” in FIGS. 30A-30D), YVEC (denoted “Y” in FIGS. 30A-30D), ornothing (Naïve, denoted “N” in FIGS. 30A-30D) as follows: 2 YU of yeastproduct were administered at 2 sites on days 0, 11, 39, 46, 60, and 67.As in the experiment above, anti-CD40 antibody was administered i.p.Nine days after the last immunization (day 76) spleens were removed,processed into single cell suspensions, and subjected to IVS for 48 hwith a mixture of recombinant HBV Surface and Core proteins (denoted“HBV Sag+Core Ag” in FIGS. 30A-30D). Supernatants were collected andevaluated by Luminex for production of IL1β, IL-6, IL-13, and IL12p70.IL-6 is a pro-inflammatory cytokine produced by antigen presenting cellsand T cells and is believed to be an important cytokine in the mechanismof action of yeast-based immunotherapeutic products. IL-13 is also apro-inflammatory cytokine produced by T cells and is closely related toIL-4 and promotion of a Th2 CD4⁺ immune response.

The results, shown in FIG. 30A (IL-1β), FIG. 30B (IL-6), FIG. 30C(IL-13) and FIG. 30D (IL-12) show that splenocytes from SCORE-immunizedmice produced IL-1β, IL-6, IL12p70, and IL-13 in response to the surfaceand core antigen mix and that the magnitude of the response was higherthan for splenocytes from YVEC-immunized or Naive mice. This antigenspecificity is consistent with results obtained for LPA in BALB/c mice(see Example 9) and for cytokine release assays in C57Bl/6 mice (seeabove).

Splenocytes from M-SPEX immunized mice produced antigen-specific signalsfor IL-1β (FIG. 30A) but not for the other cytokines. As with thefindings in C57Bl/6, this apparent difference in potency between SCOREand M-SPEX may be explained by the lower antigen content of the latter.It is expected that a-SPEX (expressing a fusion protein represented bySEQ ID NO:92, described in Example 2), which expresses higher levels ofantigen, will induce improved antigen-specific cytokine production, andin addition, IVS assays featuring the additional antigens expressed bythis product or others that incorporate other HBV antigens (HBV X andPolymerase antigens) are expected to reveal additional immunogenicity.

Example 11

The following example describes immunogenicity testing in vivo of ayeast-based immunotherapeutic composition for HBV.

In this experiment, the yeast-based immunotherapy product (GI-13002)expressing a fusion protein represented by SEQ ID NO:34 under thecontrol of the CUP1 promoter, also known as “SCORE” and morespecifically described in Example 1 was used in an adoptive transfermethod in which T cells from SCORE-immunized mice were transferred torecipient Severe Combined Immune Deficient (SCID) mice prior to tumorimplantation in the SCID mice.

Briefly, female C57BL/6 mice (age 4-6 weeks) were subcutaneouslyimmunized with GI-13002 (SCORE), YVEC (yeast containing empty vector),or nothing (naive) at 2 sites (2.5 YU flank, 2.5 YU scruff) on days 0, 7and 14. One cohort of SCORE-immunized mice was additionally injectedintraperitoneally (i.p.) with 50 μg of anti-CD40 antibody one day aftereach immunization. On day 24, mice were sacrificed and total splenocyteswere prepared and counted. Twenty-five million splenocytes in 200 μL PBSwere injected i.p. into naive recipient 4-6 week old female SCID mice.Twenty four hours post-transfer, the recipients were challengedsubcutaneously (s.c.) in the ribcage area with 300,000 SCORE-antigenexpressing EL4 tumor cells (denoted “EL-4-Score”), or tumor cellsexpressing irrelevant ovalbumin antigen. Tumor growth was monitored bydigital caliper measurement at 1 to 2 day intervals starting at day 10post tumor challenge.

The results at 10 days post tumor challenge, shown in FIG. 31,demonstrated that splenocytes from mice immunized with GI-13002 (SCORE)or GI-13002+anti-CD40 antibody, but not from YVEC or naive mice,elicited comparable protection from challenge with EL4 tumors expressingthe SCORE antigen (FIG. 31, first and second bars from left). The numberof mice with tumors 10 days post challenge are indicated above each barin FIG. 31. T cells from GI-13002-immunized mice had no effect on thegrowth of EL4 tumors expressing an unrelated antigen (not shown).Splenocytes from YVEC-immunized mice (FIG. 31, middle bar) did notaffect tumor growth, as the size and number of tumors in this group werecomparable to those of mice receiving no splenocytes (FIG. 31, far rightbar) or those mice receiving splenocytes from naïve mice (FIG. 31,second bar from right). These results indicate that immunization with ayeast-based immunotherapeutic composition expressing a surfaceantigen-core fusion protein generates an antigen-specific immuneresponse that protects SCID mice from tumor challenge. Co-administrationof the dendritic cell (DC)-activating anti-CD40 antibody did notinfluence the extent of protection.

Example 12

The following example describes the immunogenicity testing of twoyeast-based immunotherapy compositions for HBV using interferon-γ(IFN-γ) ELISpot assays.

This experiment was designed to evaluate two optimized yeast-basedimmunotherapy compositions described in Example 7 for the ability toinduce HBV antigen-specific T cells in mice immunized with thesecompositions. The experiment also tested whether novel HBV peptidesequences designed with computational algorithms and sequences obtainedfrom the published literature can be used to re-stimulate T cellresponses that were generated by these immunotherapy compositions.

In this experiment, the yeast-based immunotherapy composition describedin Example 7 as GI-13008 (“Score-C”, comprising SEQ ID NO:116) and theyeast-based immunotherapy composition described in Example 7 as GI-13013(“Spex-D”, comprising SEQ ID NO:110) were evaluated for immunogenicity.Peptide sequences used in this experiment are shown in Table 7. Thesequences denoted ZGP-5 and ZGP-7 are from the published literaturewhereas the remaining peptides were identified computationally withBIMAS or SYFPEITHI predictive algorithms. The prefixes “Db” or “Kb”refer to the haplotype of C57BL/6 mice: H-2D^(b) and H2-K^(b),respectively.

TABLE 7 Sequence MHC HBV Peptide name Amino acid sequence IdentifierClass Antigen Db9-84 WSPQAQGIL SEQ ID NO: 138 I Sag Db9-94 TVPANPPPASEQ ID NO: 141 I Sag Db9-283 GMLPVCPLL SEQ ID NO: 142 I Sag Db9-499MGLKIRQLL SEQ ID NO: 143 I Core Kb8-249 ICPGYRWM SEQ ID NO: 144 I SagKb8-262 IIFLFILL SEQ ID NO: 145 I Sag Kb8-277 VLLDYQGM SEQ ID NO: 139 ISag Kb8-347 ASVRFSWL SEQ ID NO: 140 I Sag Kb8-360 FVQWFVGLSEQ ID NO: 146 I Sag Kb8-396 LLPIFFCL SEQ ID NO: 147 I Sag ZGP-5VSFGVWIRTPPAYRPPNAPIL SEQ ID NO: 148 II Core ZGP-7 ILSPFLPLSEQ ID NO: 149 I Sag

Female C57BL/6 mice (age 4-6 weeks) were subcutaneously immunized withGI-13008 (Score-C), GI-13013 (Spex-D), YVEC (empty vector yeastcontrol), or nothing (naive) at 2 sites (2.5 YU flank, 2.5 YU scruff) ondays 0, 7 and 14. On day 20, mice were sacrificed and total splenocyteswere prepared, depleted of red blood cells, counted, and incubated at200,000 cells/well for four days in complete RPMI containing 5% fetalcalf serum plus the peptide stimulants listed in Table 7 (10 μM forD^(b) and K^(b) peptides; 30 μg/mL for ZGP peptides) or a mixture ofrecombinant HBV SAg and Core antigen (3 μg/mL total). Concanavalin A wasadded as a positive control stimulant.

The results (FIG. 32) show that immunization of C57BL/6 mice withGI-13008 (Score-C) elicits IFNγELISpot responses directed against HBVsurface (S) and core antigens with particular specificity for thefollowing peptides: Db9-84, Kb8-277 and/or Kb8-347, ZGP-5, and ZGP-7.These peptides elicited IFNγ responses greater than those from wellscontaining medium alone, or from wells containing splenocytes fromGI-13013 (Spex-D)-immunized, YVEC-immunized, or Naive mice. RecombinantS+Core antigen mixture also elicited an IFNγ response, although the YVECcontrol cells in that particular stimulant group produced backgroundsignal which precluded the evaluation of an antigen-specificcontribution for the S+Core antigen mix. These data indicate thatGI-13008 (Score-C), which expresses a surface-core fusion protein,elicits HBV-antigen specific immune responses that can be re-stimulatedwith selected peptides ex vivo, and that these responses are morereadily detectable than those elicited by GI-13013 (Spex-D).

Example 13

The following example describes an experiment in which a yeast-basedimmunotherapy composition for HBV was tested for the ability tostimulate IFNγ production from peripheral blood mononuclear cells(PBMCs) from a subject vaccinated with a commercial HBV prophylacticvaccine.

In this experiment, the yeast-based immunotherapy product known asGI-13002 (“Score”, comprising SEQ ID NO:34, Example 1) was tested forits ability to stimulate IFNγ production from PBMCs isolated from asubject who was vaccinated with commercial HBV prophylactic vaccine(ENGERIX-B®, GlaxoSmithKline), which is a prophylactic vaccinecontaining a recombinant purified hepatitis B virus surface antigen(HBsAg) adsorbed on an aluminum-based adjuvant.

Briefly, blood was collected and PBMCs were isolated from a healthyHBV-naive human subject expressing the HLA-A*0201 allele. The PBMCs werefrozen for later analysis. The subject was then vaccinated withENGERIX-B® (injection 1), blood was collected at days 12 and 29post-injection, and PBMCs were isolated and frozen. The subject wasvaccinated a second time with ENGERIX-B® (injection 2, “boost”) andblood was collected on days 10, 21, and 32 post-boost. PBMCs wereisolated and frozen for each time point.

After the series of PBMC samples was acquired and frozen, the cells fromall time points were thawed, washed, and incubated with the empty vectoryeast control (YVEC) or with GI-13002 at a 5:1 yeast:PBMC ratio for 3days in a 37° C./5% CO₂ incubator. The cells were then transferred to anIFNγELISpot plate, incubated for 18 h, and processed to develop ELISpotsaccording to standardized procedures.

As shown in FIG. 33 (columns denote time periods pre- and post-primingimmunization or post-boost), a substantial ELISpot response was observedfor GI-13002-treated PBMCs that was higher than that of YVEC-treatedPBMCs at the day 21 post-boost time-point (GI-13002 ELISpots minus YVECELISpots ˜230 spots per one million PBMCs). The level of YVEC-subtractedScore ELISpots was above the number observed for other time-points and2.8 fold above the signal obtained for the pre-vaccination sample. Theonly substantial structural difference between the yeast-basedcompositions of GI-13002 and YVEC is the presence of the surface-corefusion protein (the HBV antigen) within the vector carried by GI-13002(i.e., YVEC has an “empty” vector). Therefore, the result indicates thatGI-13002 elicited antigen-specific stimulation of T cells in the PBMCsof the subject. Because the ENGERIX-B® vaccine contains recombinantsurface antigen, but not core antigen, and because the subject wasnegative for HBV virus (had not been infected with HBV), this resultalso indicates that the IFNγ production observed was derived fromHBsAg-specific (surface antigen-specific), rather than coreantigen-specific, T cells.

Example 14

The following example describes the evaluation of yeast-basedimmunotherapy compositions for HBV in vivo in murine immunizationmodels.

In this experiment, the yeast-based immunotherapy product known asGI-13009 (“SCORE-D”, comprising SEQ ID NO:118, Example 7), and theyeast-based immunotherapy product known as GI-13020 (“X-SCORE”,comprising SEQ ID NO:130, Example 8) were administered to C57BL/6 mice,BALB/c mice and HLA-A2 transgenic mice (B6.Cg-Tg(HLA-A/H2-D)2Enge/J; TheJackson Laboratory, provided under a license from the University ofVirginia Patent Foundation). The HLA-A2 transgenic mice used in theseexperiments express an interspecies hybrid class I MHC gene, AAD, whichcontains the alpha-1 and alpha-2 domains of the human HLA-A2.1 gene andthe alpha-3 transmembrane and cytoplasmic domains of the mouse H-2D^(d)gene, under the direction of the human HLA-A2.1 promoter. The chimericHLA-A2.1/H2-D^(d) MHC Class I molecule mediates efficient positiveselection of mouse T cells to provide a more complete T cell repertoirecapable of recognizing peptides presented by HLA-A2.1 Class I molecules.The peptide epitopes presented and recognized by mouse T cells in thecontext of the HLA-A2.1/H2-D^(d) class I molecule are the same as thosepresented in HLA-A2.1⁺ humans. Accordingly, this transgenic strainenables the modeling of human T cell immune responses to HLA-A2presented antigens.

The goal of these experiments was to evaluate the breadth and magnitudeof HBV antigen-specific immune responses that are generated byimmunization with the yeast-based HBV immunotherapeutics in mice withvaried MHC alleles, including one expressing a human MHC (HLA) moleculeImmunogenicity testing was done post-immunization by ex vivo stimulationof spleen or lymph node cells with relevant HBV antigens, followed byassessment of T cell responses by: IFN-γ/IL-2 dual color ELISpot,lymphocyte proliferation assay (LPA), Luminex multi-cytokine analysis,and/or intracellular cytokine staining (ICCS). ICCS was used todetermine the contribution of CD4⁺ and CD8⁺ T cells to theantigen-specific production of IFN-γ and TNF-α.

In each of the experiments described below, mice were vaccinatedsubcutaneously with yeast-based HBV immunotherapeutics or yeast-basedcontrol (described below) according to the same regimen: injection at 2sites (flank, scruff) with 2.5 YU of the yeast composition per site,once per week for 3 weeks. Controls included YVEC (control yeastcontaining an empty vector, i.e., no antigen), OVAX2010 (a control yeastimmunotherapy composition that expresses the non-HBV antigen,ovalbumin), and the combination of YVEC with soluble recombinantantigens (ovalbumin or HBV antigens) and anti-CD40 antibody. Thespecific experiments and treatment cohorts are shown in Tables 8 and 9below. Mice were euthanized 8 days (HLA-A2 transgenic, Experiment 1) or14 days (C57BL/6 and BALB/c, Experiment 2) after the third immunization,and spleen and inguinal lymph nodes were dissected and incubated withvarious antigenic stimuli (HBV class I and class II MHC-restrictedpeptides, recombinant proteins, and HBV-antigen expressing tumor celllines) for 5 days, as indicated below. For Luminex analysis, culturesupernatants were harvested and evaluated for the production of 10different cytokines (Th1 and Th2 type) at 48 h after antigen addition.For ELISpot assays, cells were incubated on IFN-γ antibody-coated platesfor the last 24 h of the 5 day in vitro stimulation (IVS), followed bystandardized spot detection and counting. For LPAs, cells were pulsedwith ³H-thymidine for the last 18 h of the 5 day IVS, and the amount ofisotope incorporated into newly synthesized DNA was then measured byscintillation counting. For ICCS, after a full 7 day stimulation, weresubjected to Ficoll gradient centrifugation to eliminate dead cells, and1 million viable cells per well (96 well U-bottom plates) were thenincubated for 5 hours with the same antigenic stimuli at a range ofconcentrations (titration), and then permeablized, and subjected tostaining with fluorochrome coupled-antibodies recognizing intracellularIFN-γ and TNF-α plus cell surface markers CD4 and CD8. The percentage ofCD4⁺ or CD8⁺ T cells expressing the cytokines was determined by flowcytometry.

Table 8 describes the experimental cohorts and protocol forExperiment 1. In this experiment, HLA-A2 cohorts of mice were immunizedusing the protocol described above, and the mice were euthanized forimmune analysis 8 days after the third immunization. Group A (“YVEC”)received the yeast YVEC control according to the immunization scheduledescribed above; Group B (“SCORE-D (GI-13009-UL2)”) received GI-13009grown in UL2 medium (see Example 7) according to the immunizationschedule described above; and Group C (“X-SCORE (GI-13020-U2)”) receivedGI-13020 grown in U2 medium (see Example 8) according to theimmunization schedule described above.

TABLE 8 Group HLA-A2 Mice (#, treatment) A 3, YVEC B 3,SCORE-D/GI-13009-UL2 C 3, X-SCORE/GI-13020-U2

IFN-γ ELISpot assay results from the lymph node cells harvested frommice in Experiment 1 are shown in FIG. 34. This figure shows the resultsof restimulation of lymph node cells from the immunized mice withvarious HBV peptides as compared to a medium control (note that thepeptide denoted “TKO20” is a peptide from X antigen (X52-60) that iscontained within the immunotherapeutic X-SCORE, but is not present inSCORE-D). The results indicated that lymph node cells from both SCORE-D(GI-13009)-immunized and X-SCORE (GI-13020)-immunized mice possess Tcells that produce IFN-γ in response to in vitro stimulation with amixture of 3 mg/ml each of recombinant HBV surface and core antigens(FIG. 34; denoted “S&C3”). This ELISpot response was greater than thatobserved for YVEC-immunized mice (yeast controls) treated with the samestimulant, indicating that the HBV surface and/or core antigens withinthe yeast-based immunotherapeutic compositions (SCORE-D and X-SCORE) arerequired for the induction of the IFN-γ response. Furthermore, theresults indicate that the restimulation using HBV antigen in the IVSresults in efficient IFN-γ production, since wells containing mediumalone showed a much lower ELISpot response.

FIG. 34 also shows that a selected HLA-A2-restricted epitope from HBVcore (TKP16; Core:115-124 VLEYLVSFGV; SEQ ID NO:75) known in the fieldto be important in patients with acute HBV exposure and clearance,elicits a response in X-SCORE-immunized mice that is greater than thatobserved for media only wells. Further refinement of the peptideconcentration and incubation times for the ELISpot assay is expected toincrease the magnitude and reduce the variability in the observedresponse for these antigens.

FIG. 35 shows the IFN-γ ELISpot assay results from the spleen cellsharvested from SCORE-D-immunized mice in Experiment 1. The HBV corepeptide denoted “Core11-27” (ATVELLSFLPSDFFPSV (SEQ ID NO:72)) iscontained within the antigen expressed by SCORE-D, whereas the HBV Xpeptide denoted “X92-100” is not contained within the antigen expressedby SCORE-D and is therefore a control peptide in this experiment. Asshown in FIG. 35, spleen cells from the SCORE-D immunized HLA-A2transgenic mice produced an IFN-γ ELISpot response upon in vitrostimulation with the known HLA-A2 restricted HBV core epitope, denoted“Core 11-27”. This response was greater than that observed from spleencells that were stimulated in vitro with an irrelevant peptide (denoted“X92-100”), medium alone, or for any IVS treatment wells for splenocytesfrom YVEC-immunized mice.

Therefore, the initial results from Experiment 1 show that both SCORE-Dand X-SCORE elicit HBV antigen-specific T cell responses in HLA-A2transgenic mice immunized with these yeast-based immunotherapycompositions.

Table 9 describes the experimental cohorts and protocol for Experiment2. In this experiment, C57BL/6 and BALB/c cohorts of mice were immunizedusing the protocol described above, and the mice were euthanized forimmune analysis two weeks after the third immunization. Group A(“Naïve”) received no treatment; Group B (“YVEC”) received the yeastYVEC control according to the immunization schedule described above;Group C (“X-SCORE (GI-13020-U2)”) received GI-13020 grown in U2 medium(see Example 8) according to the immunization schedule described above;Group D (“SCORE-D (GI-13009-UL2)”) received GI-13009 grown in UL2 medium(see Example 7) according to the immunization schedule described above;and Group E (“OVAX2010”) received the yeast control expressing ovalbuminaccording to the immunization schedule described above.

TABLE 9 Group C57BL/6 Mice (#, treatment) BALB/c Mice (#, treatment) A8, Naïve 8, Naive B 8, YVEC 8, YVEC C 8, X-SCORE (GI-13020-U2) 8,X-SCORE (GI-13020-U2) D 8, SCORE-D (GI-13009-UL2) 8, SCORE-D(GI-13009-UL2) E 8, OVAX2010 7, OVAX2010

FIG. 36 shows the results of the ELISpot assays for lymph node cellsisolated from C57BL/6 mice immunized as indicated in Experiment 2 (Table9). These results demonstrated that both X-SCORE and SCORE-D elicitedIFN-γ responses in wild type C57BL/6 mice. Lymph node cells fromX-SCORE-immunized mice stimulated in vitro with purified, Pichiapastoris-expressed surface and core antigens (denoted yS&C; IVS with 1:1mix of Pichia-expressed surface and core antigens, 3 μg/mL each)produced a meaningful IFN-γ immune response. The same lymph node cellpreparations from both X-SCORE- and SCORE-D-immunized mice, whenstimulated with E. coli-expressed surface and core antigens (denotedcS&C; IVS with 1:1 mix of E. coli expressed surface and core antigens, 3μg/mL each), produced higher overall ELISpot responses than thoseobserved for the Pichia-expressed recombinant antigens. The columnlabeled “no stim” in FIG. 36 denotes IVS conditions where cRPMI mediumalone was provided (no antigen). In general, immunization with X-SCOREelicited a greater effect than SCORE-D, and both HBV yeast-basedimmunotherapy compositions elicited a greater response than thatobserved for YVEC-immunized (yeast control) or Naive mice. These dataindicate that both SCORE-D and X-SCORE produce HBV-antigen specificimmune responses that are detectable in ex vivo lymph node cellpreparations from C57BL/6 mice.

FIG. 37 shows the intracellular cytokine staining (ICCS) assay resultsfor C57BL/6 mice conducted in Experiment 2 (Table 9). The results showedthat immunization of C57BL/6 mice with either X-SCORE or SCORE-D elicitsIFN-γ-producing CD8⁺ T cells that are specific for the MHC Class I,H-2K^(b)-restricted peptide from surface antigen, denoted “VWL”(VWLSVIWM; SEQ ID NO:152). This effect was dependent upon theconcentration of peptide added to cells during the 5 hour incubation ofthe ICCS procedure; greater concentrations of peptide resulted in anincreasing difference in the level of IFN-γ producing CD8⁺ T cells forHBV yeast-based immunotherapeutics (SCORE-D and X-SCORE) versusirrelevant the yeast controls (Ovax and Yvec), with maximal separationoccurring at 10 μg/mL of peptide.

The ICCS assays of Experiment 2 also showed that immunization of C57BL/6mice with X-SCORE and SCORE-D elicited IFN-γ-producing CD4⁺ T cellsspecific for the MHC Class II-restricted HBV peptide from core proteindenoted “ZGP-5” (VSFGVWIRTPPAYRPPNAPIL; SEQ ID NO:148), with 0.5 μg/mLof peptide added during the 5 hour incubation period of the ICCSprocedure (FIG. 38).

Taken together with the ELISpot results described above, these dataindicate that both SCORE-D and X-SCORE yeast-based immunotherapeuticcompositions elicit HBV antigen-specific, effector CD4⁺ and CD8⁺ T cellsthat are detected by ex vivo stimulation of lymph node and spleen cellswith recombinant HBV antigens and HBV peptides. The T cell responsesoccur in both wild type C57BL/6 mice (H2-Kb) and in HLA-A2 transgenicmice, indicating the potential of these vaccines to elicit immuneresponses in the context of diverse major histocompatibility types.

Based on the results described in this Example above for HLA-A2 mice andC57BL/6 mice, as well as the results of the experiments described inExamples 9, 10, 11 and 12, it is expected that results with BALB/c miceimmunized with either SCORE-D or X-SCORE will also demonstrate that theyeast-based HBV immunotherapeutic compositions elicit HBVantigen-specific, effector CD4⁺ and CD8⁺ T cells in these mice. Indeed,initial results from the BALB/c cohorts were positive for CD8⁺ T cellresponses (data not shown). It is further expected that lymphocyteproliferation assays and Luminex cytokine release analyses will showthat both SCORE-D and X-SCORE induce immune responses specificallytargeted to the HBV antigen sequences present in the yeastimmunotherapeutics, and that these responses will be observed in allthree mouse strains (HLA-A2 transgenic, C57BL/6 and BALB/c).

Example 15

The following example describes an experiment in which yeast-basedimmunotherapy compositions for HBV are evaluated for the ability tostimulate IFNγ production from PBMCs isolated from donors of varied HBVantigen exposure.

In this experiment, the yeast-based immunotherapy product known asGI-13009 (“Score-D”, comprising SEQ ID NO:118, Example 7), and theyeast-based immunotherapy product known as GI-13020 (“X-Score”,comprising SEQ ID NO:130, Example 8) are tested for their ability tostimulate IFNγ production from PBMCs isolated from donors of varied HBVantigen exposure. In this experiment, one group of donors has previouslybeen vaccinated with ENGERIX-B® (GlaxoSmithKline) or with RECOMBIVAX HB®(Merck & Co., Inc.), one group of donors is naïve to HBV antigen(“normal”), and one group of donors is a chronic HBV patient (a subjectchronically infected with HBV). ENGERIX-B® is a prophylactic recombinantsubunit vaccine containing a recombinant purified hepatitis B virussurface antigen (HBsAg) produced in yeast cells, purified and thenadsorbed on an aluminum-based adjuvant. RECOMBIVAX HB® is a prophylacticrecombinant subunit vaccine derived from HBV surface antigen (HBsAg)produced in yeast cells and purified to contain less than 1% yeastprotein. All donors express the HLA-A*0201 allele.

The donor PBMCs are incubated in 6-well flat-bottomed tissue cultureplates (10⁷ PBMCs per well) for 3 h in a 5% CO₂ incubator in completeRPMI medium containing 10% fetal bovine serum. Non-adherent cells areremoved and discarded and the adherent cells are treated withrecombinant human interleukin-4 (IL-4) and recombinant human granulocytemacrophage colony-stimulating factor (GM-CSF) (20 and 50 ng/mL,respectively) for 5 days to generate immature dendritic cells (iDCs).The iDCs are then incubated with anti-CD40 antibody (1 μg/ml), YVEC(yeast control comprising an empty vector), or the yeast-based productsGI-13020 or GI-13009, for 48 h in a 5% CO₂ incubator at 37° C., togenerate mature DCs. For anti-CD40 antibody-treated DCs, cells areadditionally pulsed with HLA-A*0201-restricted HBV peptides usingstandard methods. All DC groups are PBS-washed and then removed fromplates with a cell harvester in PBS. Cells are irradiated (30 Gy) andused to stimulate the autologous donor PBMCs at a DC:PBMCs ratio of1:10. Stimulation is conducted for 7 days (round 1) of which the last 4days are conducted in medium containing recombinant human IL-2. Thestimulated PBMCs are then subjected to Ficoll gradient centrifugation,and the isolated viable cells subjected to a second round of IVS withyeast-pulsed or peptide-pulsed DCs prepared as described above. Thestimulated PBMCs are then incubated with HBV peptide(s) or controls inthe presence of 20 U/mL rhIL-2 in 96 well plates coated with antibodyspecific for IFN-γ, and ELISpot detection is conducted using standardmanufacturer procedures. It is expected that PBMCs stimulated withautologous SCORE-D- or X-SCORE-fed DCs, or with HBV peptide-pulsed DCs,will respond to exogenous HBV peptides to a greater degree than PBMCsstimulated with YVEC-fed or unpulsed DCs, and that this effect will bemore pronounced for HBV ENGERIX® or RECOMBIVAX HB® vaccine recipientsthan for donors who are naive to HBV antigen exposure.

Example 16

The following example describes preclinical experiments using humanPBMCs to demonstrate the immunogenicity of yeast-based HBV immunotherapycompositions of the invention in humans.

Specifically, these experiments are designed to determine whether HBVsurface antigen-specific and/or HBV core antigen-specific CD8⁺ T cellscan be detected in the peripheral blood mononuclear cells (PBMCs) of HBVcarriers following 2 rounds of in vitro stimulation (IVS) withyeast-based HBV immunotherapy compositions containing HBV surfaceantigen and HBV core.

PBMCs are obtained from human donors confirmed to be positive for HBV(based on serum HBsAg status). Total DNA is isolated from 0.5 mL wholeblood and typed for HLA in order to identify the correct HBV pentamerfor testing (see table below).

TABLE 10 HLA type Pentamer Peptide Sequence Antigen A*0201FLLTRILTI (SEQ ID NO: 42) Surface A*0201 GLSPTVWLSV (SEQ ID NO: 43)Surface A*0201 FLPSDFFPSI (SEQ ID NO: 44) Core A*1101YVNVNMGLK (SEQ ID NO: 48) Core A*2402 EYLVSFGVW (SEQ ID NO: 49) Core

Dendritic cells (DCs) are prepared from the PBMCs isolated from thedonors described above by culturing PBMCs for 5 days in the presence ofGM-CSF and IL-4. The DCs are subsequently incubated with yeast-based HBVimmunotherapy compositions (e.g., those described in any of Examples 1-8or elsewhere herein) or control yeast (e.g., “YVEC”, which isSaccharomyces cerevisiae yeast that is transformed with an empty vector,or vector that does not contain an antigen-encoding insert), at a ratioof 1:1 (yeast:DCs). Control DC cultures also include DCs incubated withHBV peptides, control peptides (non-HBV peptides), or nothing.

After 48-hours in co-culture, the DCs are used as antigen presentingcells (APCs) for stimulation of autologous T cells (i.e., T cells fromthe donors). Each cycle of stimulation, designated as IVS (in vitrostimulation), consists of 3 days culture in the absence of IL-2,followed by 4 additional days in the presence of recombinant IL-2 (20U/ml). At the end of IVS 2, T cells are stained with a control tetrameror pentamer or a tetramer or pentamer specific for an HBV peptideepitope identified above. The percentage of CD8⁺ T cells that stainpositive with the tetramer or pentamer is quantified by flow cytometry.

It is expected that stimulation of donor T cells from HBV-positivedonors with a yeast-HBV immunotherapeutic of the invention increases thepercentage of tetramer/pentamer-positive CD8⁺ T cells in at least someor a majority of the donors, as compared to controls, indicating thathuman T cells from HBV-infected individuals have the capacity torecognize HBV proteins carried by the yeast-based immunotherapy asimmunogens.

Additional experiments similar to those above are run using donor PBMCsfrom normal (non-HBV infected) individuals. It is expected thatstimulation of donor T cells from normal donors with a yeast-HBVimmunotherapeutic of the invention increases the percentage oftetramer/pentamer-positive CD8⁺ T cells in at least some or a majorityof the donors, as compared to controls, indicating that human T cellsfrom non-infected individuals also have the capacity to recognize HBVproteins carried by the yeast-based immunotherapy as immunogens.

In an additional experiment, HBV-specific T cells from three of thedonors from the experiments described above are expanded in vitro usingDCs incubated with HBV yeast-based immunotherapeutics (e.g., thosedescribed in any of Examples 1-8 or elsewhere herein) for 2 cycles ofIVS (as described above). A third IVS is carried out with DCs matured inpresence of CD40L and pulsed with the HBV peptide(s). At day 5, CD8⁺ Tcells are isolated and used in an overnight cytotoxic T lymphocyte (CTL)assay against tumor cell targets expressing HBV antigens, at variouseffector:target (ET) ratios. The percentage of CD8⁺ T cells that stainpositive with a control tetramer/pentamer versus an HBV-specifictetramer/pentamer is measured.

It is expected that T cells from some or all of the donors will becapable of generating CD8⁺ CTLs that can kill targets expressing HBVantigens. These data will demonstrate that yeast-HBV immunotherapeuticcompositions can generate HBV-specific CTLs that are capable of killingan HBV antigen-expressing tumor cell.

Example 17

The following example describes a phase 1 clinical trial in healthyvolunteers.

A 12-week, open-label dose escalation phase 1 clinical study isperformed using a yeast-based HBV immunotherapy composition describedherein as GI-13009 (“SCORE-D”, comprising SEQ ID NO:118, Example 7), oralternatively, the yeast-based HBV immunotherapy composition describedherein as GI-13020 (“X-SCORE”, comprising SEQ ID NO:130, Example 8) isused. Other yeast-based HBV immunotherapy compositions described herein(e.g., any of those described in Examples 1-8) can be utilized in asimilar phase 1 clinical trial. The yeast-based HBV immunotherapyproduct for the phase 1 clinical trial is selected from pre-clinicalstudies (e.g., those described in any one of Examples 9-17) on the basisof considerations including strongest net immune response profile (e.g.,amplitude of response for T cell epitopes that are most predictive ofpositive outcome, and/or breadth of immune response across the range ofepitopes).

Subjects are immune active and healthy volunteers with no prior orcurrent indication or record of HBV infection.

Approximately 48 subjects (6 arms, 8 subjects per arm) meeting thesecriteria are administered the yeast-based HBV immunotherapy compositionin a sequential dose cohort escalation protocol utilizing one of twodifferent dosing protocols as follows:

Protocol A: Prime-Boost Dosing (4 weekly doses starting at Day 1,followed by 2 monthly doses at Week 4 & Week 8)

Arm 1A: 20 Y.U. (administered in 10 Y.U. doses to 2 different sites);

Arm 2A: 40 Y.U. (administered in 10 Y.U. doses to 4 different sites);

Arm 3A: 80 Y.U. (administered in 20 Y.U. doses to 4 different sites)

4-Weekly Dosing (three total doses administered at Day 1, Week 4 andWeek 8)

Arm 1B: 20 Y.U. (administered in 10 Y.U. doses to 2 different sites);

Arm 2B: 40 Y.U. (administered in 10 Y.U. doses to 4 different sites);

Arm 3B: 80 Y.U. (administered in 20 Y.U. doses to 4 different sites)

All doses are administered subcutaneously and the dose is divided amongtwo or four sites on the body (every visit) as indicated above. Safetyand immunogenicity (e.g., antigen-specific T cell responses measured byELISpot and T cell proliferation) are assessed. Specifically, anELISpot-based algorithm is developed for categorical responders. ELISpotassays measuring regulatory T cells (Treg) are also assessed and CD4⁺ Tcell proliferation in response to HBV antigens is assessed andcorrelated with the development of anti-Saccharomyces cerevisiaeantibodies (ASCA).

It is expected that the yeast-based HBV immunotherapeutic will bewell-tolerated and show immunogenicity as measured by one or more ofELISpot assay, lymphocyte proliferation assay (LPA), ex vivo T cellstimulation by HBV antigens, and/or ASCA.

Example 18

The following example describes a phase 1b/2a clinical trial in subjectschronically infected with hepatitis B virus.

Due to a tendency of HBV infected patients to experience destabilizingexacerbations of hepatitis as part of the natural history of thedisease, yeast-based HBV immunotherapy is initiated after some period ofpartial or complete virologic control using anti-viral-based therapy,with a primary efficacy goal of improving seroconversion rates. In thisfirst consolidation approach, yeast-based HBV immunotherapy is used inpatients after they achieve HBV DNA negativity by PCR to determinewhether seroconversion rates can be improved in combination withcontinued anti-viral therapy.

An open-label dose escalation phase 1b/2a clinical trial is run using ayeast-based HBV immunotherapy composition described herein as GI-13009(“SCORE-D”, comprising SEQ ID NO:118, Example 7), or alternatively, theyeast-based HBV immunotherapy composition described herein as GI-13020(“X-SCORE”, comprising SEQ ID NO:130, Example 8) is used. Otheryeast-based HBV immunotherapy compositions described herein (e.g., anyof those described in Examples 1-8) can be utilized in a similar phase 1clinical trial. Subjects are immune active and chronically infected withhepatitis B virus (HBV) that is well controlled by anti-viral therapy(i.e., tenofovir disoproxil fumarate, or TDF (VIREAD®)) as measured byHBV DNA levels. Subjects are negative for HBV DNA (below detectablelevels by PCR or <2000 IU/ml), but to qualify for this study, subjectsmust be HBeAg positive and have no evidence of cirrhosis ordecompensation.

In stage one of this study, approximately 40 subjects (˜5 subjects perarm) meeting these criteria are administered the yeast-based HBVimmunotherapy composition in a sequential dose cohort escalationprotocol utilizing dose ranges from 0.05 Y.U. to 80 Y.U. (e.g., 0.05Y.U., 10 Y.U., 20 Y.U., and 40-80 Y.U.). In one protocol, 5 weekly doseswill be administered subcutaneously (weekly dosing for 4 weeks),followed by 2-4 monthly doses also administered subcutaneously, withcontinued anti-viral therapy during treatment with the yeast-based HBVimmunotherapy (prime-boost protocol). In a second protocol, a 4-weeklydosing protocol is followed, where subjects receive a total of threedoses administered on day 1, week 4 and week 8, using the sameescalating dose strategy as set forth above. Optionally, in one study, asingle patient cohort (5-6 patients) will receive subcutaneousinjections of placebo (PBS) on the same schedule as the immunotherapyplus continued anti-viral therapy. Conservative stopping rules are inplace for ALT flares and signs of decompression.

In the second stage of this trial, subjects (n=60) are randomized 30 perarm to continue on anti-viral (TDF) alone or anti-viral plus theyeast-based HBV immunotherapeutic protocol (dose 1 and dose 2) for up to48 weeks.

Safety, HBV antigen kinetics, HBeAg and HBsAg seroconversion, andimmunogenicity (e.g., antigen-specific T cell responses measured byELISpot) are assessed. In addition, dose-dependent biochemical (ALT) andviral load is monitored. Specifically, measurement of serum HBsAgdecline during treatment between the 3-treatment arms at weeks 12, 24,48 is measured, and HBsAg-loss/seroconversion is measured at week 48.

An increase in rates of HBsAg loss and/or seroconversion to >20% at 48weeks in subjects receiving the yeast-based immunotherapy and TDF, ascompared to subjects receiving TDF alone, is considered a clinicallymeaningful advancement. The yeast-based HBV immunotherapy composition isexpected to provide a therapeutic benefit to chronically infected HBVpatients. The immunotherapy is expected to be safe and well-tolerated atall doses delivered. Patients receiving at least the highest dose ofyeast-based HBV immunotherapy are expected to show treatment-emergent,HBV-specific T cell responses as determined by ELISPOT, and patientswith prior baseline HBV-specific T cell responses are expected to showimproved HBV-specific T cell responses while on treatment. Patientsreceiving yeast-based HBV immunotherapy are expected to show improvementin seroconversion rates as compared to the anti-viral group and/or ascompared to the placebo controlled group, if utilized. Improvements inALT normalization are expected in patients receiving yeast-based HBVimmunotherapy.

In an alternate trial, HBeAg negative patients meeting the othercriteria (immune active, chronically HBV infected, well-controlled onanti-virals, with no signs of decompensation) are treated in a similardose escalation trial as described above (or at the maximum tolerateddose or best dose identified in the trial described above). Patients aremonitored for safety, immunogenicity, and HBsAg seroconversion.

Example 19

The following example describes a phase 1b/2a clinical trial in subjectschronically infected with hepatitis B virus.

An open-label dose escalation phase 1b/2a clinical trial is run using ayeast-based HBV immunotherapy composition described herein as GI-13009(“SCORE-D”, comprising SEQ ID NO:118, Example 7), or alternatively, theyeast-based HBV immunotherapy composition described herein as GI-13020(“X-SCORE”, comprising SEQ ID NO:130, Example 8) is used. Otheryeast-based HBV immunotherapy compositions described herein (e.g., anyof those described in Examples 1-8) can be utilized in a similar phase1b/2a clinical trial. Subjects are immune active and chronicallyinfected with hepatitis B virus (HBV) that has been controlled byanti-viral therapy (e.g. tenofovir (VIREAD®)) for at least 3 months.Subjects are not required to have completely cleared the virus to enrollin the study, i.e., patients may be positive or negative for HBV DNA(negativity determined as below detectable levels by PCR or <2000IU/ml); however, to qualify for this study, subjects have no evidence ofcirrhosis or decompensation. Patients may be HBeAg-positive, althoughHBeAg-negative patients can be included in the study.

30-40 subjects (6-10 patients per cohort) meeting these criteria areadministered the yeast-based HBV immunotherapy composition in asequential dose cohort escalation protocol utilizing dose ranges from0.05 Y.U. to 40 Y.U. (e.g., 0.05 Y.U., 0.5 Y.U., 4 Y.U., 40 Y.U.), orutilizing dose ranges from 0.05 Y.U. to 80 Y.U. (e.g., 0.05 Y.U., 10Y.U., 20 Y.U., 40/80 Y.U.). In one protocol, 5 weekly doses will beadministered subcutaneously (weekly dosing for 4 weeks), followed by 2-4monthly doses also administered subcutaneously, with continuedanti-viral therapy during treatment with the yeast-based HBVimmunotherapy (prime-boost protocol). In a second protocol, a 4-weeklydosing protocol is followed, where subjects receive a total of threedoses administered on day 1, week 4 and week 8, using the sameescalating dose strategy as set forth above. In one study, a singlepatient cohort (5-6 patients) will receive subcutaneous injections ofplacebo (PBS) on the same schedule as the immunotherapy plus continuedanti-viral therapy. Conservative stopping rules are in place for ALTflares and signs of decompression.

Safety, HBeAg and HBsAg seroconversion, viral control (e.g., developmentof viral negativity or trend toward viral negativity), andimmunogenicity (e.g., antigen-specific T cell responses measured byELISpot) are assessed. In addition, dose-dependent biochemical (ALT) andviral load is monitored.

>1 log 10 reduction in HB-SAg by 24 weeks or >1 log 10 reduction inHB-eAg by 12 weeks are considered to be endpoints for phase 2a. For HBVseroconversion, an SAg seroconversion of 10% by 24 weeks, and 15% by 48weeks, and/or an eAg seroconversion rate of 25% by 24 weeks or 50% by 48weeks are success criteria.

The yeast-based HBV immunotherapy composition is expected to provide atherapeutic benefit to chronically infected HBV patients. Theimmunotherapy is expected to be safe and well-tolerated at all dosesdelivered. Patients receiving at least the highest dose of yeast-basedHBV immunotherapy are expected to show treatment-emergent, HBV-specificT cell responses as determined by ELISPOT and patients with priorbaseline HBV-specific T cell responses show improved HBV-specific T cellresponses while on treatment. Patients receiving yeast-based HBVimmunotherapy will show improvement in seroconversion rates as comparedto available comparative data for the given anti-viral and/or ascompared to the placebo controlled group. Patients receiving yeast-basedHBV immunotherapy will show improvement in viral loss (e.g., viralnegativity as measured by PCR). Improvements in ALT normalization areexpected in patients receiving yeast-based HBV immunotherapy.

Example 20

The following example describes a phase 2 clinical trial in subjectschronically infected with hepatitis B virus.

A randomized phase 2 clinical trial in patients chronically infectedwith HBV treats treatment-naïve, HBeAg-positive (and possiblyHBeAg-negative) subjects with ALT>2×ULN and viral loads >1 millioncopies. The subjects (˜60 subjects per arm adjusted based on phase 1study signal) must have at least 6 months of prior anti-viral therapy,and have viral negativity for 2 consecutive visits at least one monthapart. Subjects are randomized into two arms. Arm 1 patients receive24-48 weeks of yeast-based HBV immunotherapy (e.g., yeast-based HBVimmunotherapy composition described herein as GI-13009 (“SCORE-D”,comprising SEQ ID NO:118, Example 7), or alternatively, the yeast-basedHBV immunotherapy composition described herein as GI-13020 (“X-SCORE”,comprising SEQ ID NO:130, Example 8). All patients receivingimmunotherapy continue anti-viral therapy (e.g., tenofovir (VIREAD®)).Arm 2 patients receive a placebo (PBS control injection) with continuedanti-viral therapy. The primary endpoint is seroconversion and viralnegativity. Additional yeast-based HBV immunotherapy compositionsdescribed herein (e.g., any of those described in Examples 1-8) can alsobe utilized in a phase 2 trial with similar design.

Patients who achieve seroconversion receive 6-12 month consolidationtherapy on either yeast-immunotherapy and antivirals (Arm 1) orantivirals alone (Arm 2), followed by a 6 month treatment holiday. Thenumber of patients remaining in remission after completion of the 6month holiday represent the secondary endpoint of the study. Additionalendpoints include safety, immunogenicity and ALT normalization, asdiscussed in the Examples describing human clinical trials above.

The yeast-based HBV immunotherapy composition is expected to provide atherapeutic benefit to chronically infected HBV patients. Theimmunotherapy is expected to be safe and well-tolerated. Patientsreceiving yeast-based HBV immunotherapy are expected to showtreatment-emergent, HBV-specific T cell responses as determined byELISPOT and patients with prior baseline HBV-specific T cell responsesshow improved HBV-specific T cell responses while on treatment. Patientsreceiving yeast-based HBV immunotherapy are expected to show animprovement in seroconversion rates as compared to the placebocontrolled group. Patients receiving yeast-based HBV immunotherapy areexpected to show an improvement in viral loss (e.g., viral negativity asmeasured by PCR). Improvements in ALT normalization are expected inpatients receiving yeast-based HBV immunotherapy.

While various embodiments of the present invention have been describedin detail, it is apparent that modifications and adaptations of thoseembodiments will occur to those skilled in the art. It is to beexpressly understood, however, that such modifications and adaptationsare within the scope of the present invention, as set forth in thefollowing claims.

What is claimed is:
 1. A recombinant nucleic acid molecule encoding afusion protein comprising HBV antigens, wherein the HBV antigens consistof: i) an HBV X antigen having an amino acid sequence that is at least95% identical to positions 1-60 of SEQ ID NO:130; ii) an HBV surfaceantigen having an amino acid sequence that is at least 95% identical topositions 63-461 of SEQ ID NO:130; and iii) an HBV core antigen havingan amino acid sequence that is at least 95% identical to positions 462to 643 of SEQ ID NO:130.
 2. The recombinant nucleic acid molecule ofclaim 1, wherein the recombinant nucleic acid molecule comprises anucleic acid sequence of SEQ ID NO:129.
 3. An isolated cell transfectedwith the recombinant nucleic acid molecule of claim
 1. 4. The isolatedcell of claim 3, wherein the cell is a yeast cell.
 5. The recombinantnucleic acid molecule of claim 1, wherein the fusion protein comprisesan amino acid sequence that is at least 95% identical to the amino acidsequence of SEQ ID NO:130.
 6. The recombinant nucleic acid molecule ofclaim 1, wherein the fusion protein comprises an amino acid sequencethat is at least 95% identical to the amino acid sequence of SEQ IDNO:150.
 7. The recombinant nucleic acid molecule of claim 1, wherein thefusion protein comprises the amino acid sequence of SEQ ID NO:130 or SEQID NO:150.
 8. The recombinant nucleic acid molecule of claim 1, whereinthe fusion protein consists of an amino sequence of SEQ ID NO:130. 9.The recombinant nucleic acid molecule of claim 1, wherein the fusionprotein consists of an amino sequence of SEQ ID NO:150.
 10. An isolatedcell transfected with the recombinant nucleic acid molecule of claim 8.11. The isolated cell of claim 10, wherein the cell is a yeast cell. 12.An isolated cell transfected with the recombinant nucleic acid moleculeof claim
 9. 13. The isolated cell of claim 12, wherein the cell is ayeast cell.
 14. The recombinant nucleic acid molecule of claim 1,wherein the amino acid sequence of HBV X antigen consists of positions1-60 of SEQ ID NO:130.
 15. The recombinant nucleic acid molecule ofclaim 1, wherein the amino acid sequence of HBV surface antigen consistsof positions 63-461 of SEQ ID NO:130.
 16. The recombinant nucleic acidmolecule of claim 1, wherein the amino acid sequence of HBV core antigenconsists of positions 462 to 643 of SEQ ID NO:130.